Genetically engineered hematopoietic stem cells and uses thereof

ABSTRACT

Genetically engineered hematopoietic cells such as hematopoietic stem cells having one or more genetically edited genes of lineage-specific cell-surface proteins and therapeutic uses thereof, either alone or in combination with immune therapy that targets the lineage-specific cell-surface proteins.

This application is a continuation of U.S. Ser. No. 17/176,610, filedFeb. 16, 2021, which is a continuation of U.S. Ser. No. 16/701,818, nowU.S. Pat. No. 10,925,902, filed Dec. 3, 2019, which is a continuation ofU.S. Ser. No. 16/554,520, filed Aug. 28, 2019, which claims priority toU.S. Ser. No. 62/723,993 filed Aug. 28, 2018, U.S. Ser. No. 62/728,061filed Sep. 6, 2018, U.S. Ser. No. 62/789,440 filed Jan. 7, 2019, andU.S. Ser. No. 62/809,202 filed Feb. 22, 2019, the entire contents ofeach of which is incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 7, 2021, isnamed V029170002US11-SUBSEQ-CEW and is 93,686 bytes in size.

BACKGROUND OF THE INVENTION

A major challenge in designing targeted therapies is the successfulidentification of proteins that are uniquely expressed on cells thatwould be therapeutically relevant to eliminate (e.g., abnormal,malignant, or other target cells) but not present on cells that one doesnot wish to eliminate (e.g., normal, healthy, or other non-targetcells). For example, many cancer therapeutics struggle to effectivelytarget cancer cells while leaving normal cells unharmed.

An alternative strategy that has emerged involves targeting an entirecell lineage, which includes targeting normal cells, cancer cells, andpre-cancerous cells. For example, CD19-targeted chimeric antigenreceptor T cells (CAR T cells) and anti-CD20 monoclonal antibodies (e.g.Rituximab) each target B cell lineage proteins (CD19 and CD20,respectively). While potentially effective in treating B cellmalignancies, use of such therapies is limited as elimination of B cellsis detrimental. Similarly, targeting lineage-specific proteins of othercell populations, for example, myeloid lineage cells (e.g., cancersarising from myeloid blasts, monocytes, megakaryocytes, etc.) is notfeasible, as these cell populations are necessary for survival.

Thus, there remains an unmet need to effectively target cells ofinterest, e.g., cancer cells, without targeting or harming normal cellpopulations.

SUMMARY OF THE INVENTION

Provided herein are compositions, e.g., engineered cells, and methodsthat provide the ability to target one or more cells or cell populationsof interest while allowing non-targeted cell populations to escape suchtargeting. For example, provided herein are genetically engineeredhematopoietic cells such as hematopoietic stem cells (HSCs) havinggenetically modified or edited genes of one or more lineage-specificcell-surface antigens. In some embodiments, the modified, e.g., editedgenes are able to produce the lineage-specific cell surface proteins inmodified form, which retain, at least partially, the biological activityof the lineage-specific cell-surface antigens in the HSCs or indescendant cells expressing such, but can escape targeting by cytotoxicagents that are specific to the wild-type lineage-specific cell-surfaceantigens. In some embodiments, the modified, e.g., edited, genes do notproduce the lineage-specific cell surface protein(s) or produce atruncated version of the lineage-specific cell surface protein(s) that,while able to escape targeting by cytotoxic agents that are specific tothe wild-type lineage-specific cell-surface antigen(s), may not retainbiological activity of the lineage-specific cell-surface antigen(s) inthe HSCs or in descendant cells expressing such.

Thus, provided herein are genetically engineered hematopoietic cells,such as hematopoietic stem cells (HSCs), having one or more modifiedlineage-specific cell-surface antigen. In some embodiments, the one ormore modified lineage-specific cell surface proteins are modified suchthat one or more of the lineage-specific cell surface proteins retain atleast partially its biological activity of the lineage-specificcell-surface antigens in the HSCs or in descendant cells expressingsuch, but can escape targeting by cytotoxic agents that are specific tothe corresponding wild-type lineage-specific cell-surface antigen(s). Insome embodiments, the one or more modified lineage-specific cell surfaceproteins are modified such that all of the modified lineage-specificcell surface proteins (e.g., one, two, three, four, etc.) retain atleast partial biological activity. In some embodiments, the one or moremodified lineage-specific cell surface proteins are modified such thatat least one, but not all, of the modified lineage-specific cell surfaceprotein(s) retain at least partial biological activity. In someembodiments, the one or more modified lineage-specific cell surfaceproteins are modified such that one or more of the lineage-specific cellsurface proteins do not retain at least partial biological activity ofthe lineage-specific cell-surface antigens in the HSCs or in descendantcells expressing such, but can escape targeting by cytotoxic agents thatare specific to the corresponding wild-type lineage-specificcell-surface antigen(s). In some embodiments, the one or more modifiedlineage-specific cell surface proteins are modified such that none ofthe modified lineage-specific cell surface protein(s) retain at leastpartial biological activity. Thus, the genetically engineeredhematopoietic cells provided herein having one or more modifiedlineage-specific cell-surface antigens can escape targeting by cytotoxicagents that are specific to the corresponding wild-type lineage-specificcell-surface antigen(s) and may comprise modified lineage-specificcell-surface antigen(s) that retain at least partial biological activityand/or may comprise lineage-specific cell-surface antigen(s) that do notretain biological activity (e.g., the protein may be knocked out).

The genetically engineered hematopoietic cells provided herein havinggenetically modified or edited genes of one or more lineage-specificcell-surface antigens are useful in therapies, e.g., immunotherapies andother cytotoxic agents, that specifically target cells expressing alineage-specific cell-surface antigen, by virtue of the fact that thegenetically engineered hematopoietic cells produce one or more modifiedlineage-specific cell-surface antigen(s) that are able to escape suchtargeting while retaining their biological activity. Accordingly, withsuch engineered hematopoietic cell it is possible to target or directimmunotherapies or other cytotoxic agents against a lineage-specificcell-surface antigen that is required for survival of an organism. Also,with such engineered hematopoietic cell, it is possible to target ordirect immunotherapies or other cytotoxic agents against a cell typerequired for survival of an organism expressing a targetedlineage-specific cell-surface antigen. In other embodiments, thegenetically engineered hematopoietic cells provided herein havinggenetically modified or edited genes of one or more lineage-specificcell-surface antigens are useful in therapies, e.g., immunotherapies andother cytotoxic agents, that specifically target cells expressing alineage-specific cell-surface antigen, by having the ability to escapesuch targeting even though that do not retain biological activity. Withsuch engineered hematopoietic cell it is possible to target or directimmunotherapies or other cytotoxic agents against a lineage-specificcell-surface antigen that is not required for survival of an organism.Also, with such engineered hematopoietic cell, it is possible to targetor direct immunotherapies or other cytotoxic agents against a cell typethat is not required for survival of an organism expressing a targetedlineage-specific cell-surface antigen. In some embodiments, in which thegenetically engineered hematopoietic cells have one or more modifiedlineage-specific cell-surface antigens, wherein one or morelineage-specific cell-surface antigens retain biological activity andwherein one or more lineage-specific cell-surface antigens do not retainbiological activity, it is possible to target or direct immunotherapiesor other cytotoxic agents against lineage-specific cell-surfaceantigen(s) that may or may not be required for survival of an organism.Also, with such engineered hematopoietic cell, it is possible to targetor direct immunotherapies or other cytotoxic agents against a cell typethat may or may not be required for survival of an organism expressing atargeted lineage-specific cell-surface antigen.

In some aspects, the genetically engineered hematopoietic cell isgenetically modified or edited such that it produces one modified ormutated lineage-specific cell-surface antigen that retains biologicalactivity, but escapes targeting by a cytotoxic agent specific to thewild-type lineage-specific cell-surface antigen. In some aspects, thegenetically engineered hematopoietic cell is genetically modified oredited such that it produces two or more (e.g., 2, 3, 4, 5, etc.)modified or mutated lineage-specific cell-surface antigens that escapetargeting by a cytotoxic agent specific to (or that targets) thecorresponding wild-type lineage-specific cell-surface antigens. In someembodiments of these latter aspects, at least one of the modified ormutated lineage-specific cell-surface proteins retains its biologicalactivity. In some embodiments, two or more of the modified or mutatedlineage-specific cell-surface antigens retain their respectivebiological activities. In some embodiments, all of the modified ormutated lineage-specific cell-surface antigens expressed in thegenetically engineered hematopoietic cell retain (at least partially)their respective biological activities.

Accordingly, one aspect of the present disclosure features a geneticallyengineered hematopoietic cell, comprising: (i) a first gene encoding afirst lineage-specific cell-surface antigen, which gene has beenmodified or edited and (ii) a second gene encoding a secondlineage-specific cell-surface antigen, which gene has been modified oredited. In some embodiments, the first gene has been modified or editedsuch that expression of the first lineage-specific cell-surface antigenis reduced or eliminated in the genetically engineered hematopoieticcell (e.g., as compared with expression of the corresponding endogenousor wild-type lineage-specific cell-surface antigen). In someembodiments, the first gene has been modified or edited such that thegenetically engineered hematopoietic cell expresses a mutant or modifiedversion of the first lineage-specific cell-surface antigen. In someembodiments, the mutant or modified version of the firstlineage-specific cell-surface antigen escapes targeting by a cytotoxicagent that targets the corresponding wild-type lineage-specificcell-surface antigen. In some embodiments, the mutant or modifiedversion of the first lineage-specific cell-surface antigen retains itsbiological activity. In some embodiments, the mutant or modified versionof the first lineage-specific cell-surface antigen escapes targeting bya cytotoxic agent that targets the corresponding wild-typelineage-specific cell-surface antigen and retains its biologicalactivity. In some embodiments, the second gene has been modified oredited such that expression of the second lineage-specific cell-surfaceantigen is reduced or eliminated in the genetically engineeredhematopoietic cell (e.g., as compared with expression of thecorresponding endogenous or wild-type lineage-specific cell-surfaceantigen). In some embodiments, the second gene has been modified oredited such that the genetically engineered hematopoietic cell expressesa mutant or modified version of the second lineage-specific cell-surfaceantigen. In some embodiments, the mutant or modified version of thesecond lineage-specific cell-surface antigen escapes targeting by acytotoxic agent that targets the corresponding wild-typelineage-specific cell-surface antigen. In some embodiments, the mutantor modified version of the second lineage-specific cell-surface antigenretains its biological activity. In some embodiments, the mutant ormodified version of the second lineage-specific cell-surface antigenescapes targeting by a cytotoxic agent that targets the correspondingwild-type lineage-specific cell-surface antigen and retains itsbiological activity. In some embodiments, the mutant or modifiedversions of the first and second lineage-specific cell-surface antigensretain their respective biological activities. In some embodiments, themutant or modified version of the first lineage-specific cell-surfaceantigen retains its biological activity and the mutant or modifiedversion of the second lineage-specific cell-surface antigen does notretain its biological activity. In some embodiments, neither the mutantor modified version of the first lineage-specific cell-surface antigenretains its biological activity nor the mutant or modified version ofthe second lineage-specific cell-surface antigen retains its biologicalactivity.

Another aspect of the present disclosure features a geneticallyengineered hematopoietic cell, comprising: (i) a first gene encoding afirst lineage-specific cell-surface antigen, which gene has beenmodified or edited; (ii) a second gene encoding a secondlineage-specific cell-surface antigen, which gene has been modified oredited; and (iii) a third gene encoding a third lineage-specificcell-surface antigen, which gene has been modified or edited. In someembodiments, the genetically engineered hematopoietic cell furthercomprises (iv) a fourth gene encoding a fourth lineage-specificcell-surface antigen, which gene has been modified or edited. In someembodiments, the genetically engineered hematopoietic cell furthercomprises (v) a fifth gene encoding a fifth lineage-specificcell-surface antigen, which gene has been modified or edited.

In some embodiments, any one or more of the gene(s) encoding alineage-specific cell-surface antigen has been modified or edited suchthat expression of the respective lineage-specific cell-surfaceantigen(s) is reduced or eliminated in the genetically engineeredhematopoietic cell (e.g., as compared with expression of thecorresponding endogenous or wild-type lineage-specific cell-surfaceantigen). In some embodiments, any one or more of the gene(s) encoding alineage-specific cell-surface antigen has been modified or edited suchthat the genetically engineered hematopoietic cell expresses a mutant ormodified version of the respective lineage-specific cell-surfaceantigen(s).

In some embodiments, any one or more of the mutant lineage-specificcell-surface antigen(s) escapes targeting by a cytotoxic agent thattargets the corresponding wild-type lineage-specific cell-surfaceantigen. In some embodiments, any one or more of the mutantlineage-specific cell-surface antigen(s) retains its biologicalactivity. In some embodiments, any one or more of the mutantlineage-specific cell-surface antigen(s) escapes targeting by acytotoxic agent that targets the corresponding wild-typelineage-specific cell-surface antigen and retains its biologicalactivity.

Another aspect of the present disclosure features a population ofgenetically engineered hematopoietic cells, wherein the geneticallyengineered hematopoietic cells in the population comprise: (i) a firstgene encoding a first lineage-specific cell-surface antigen, which genehas been modified or edited and (ii) a second gene encoding a secondlineage-specific cell-surface antigen, which gene has been modified oredited. In some embodiments, the first gene has been modified or editedsuch that expression of the first lineage-specific cell-surface antigenis reduced or eliminated in the genetically engineered hematopoieticcell (e.g., as compared with expression of the corresponding endogenousor wild-type lineage-specific cell-surface antigen). In someembodiments, the first gene has been modified or edited such that thegenetically engineered hematopoietic cell expresses a mutant or modifiedversion of the first lineage-specific cell-surface antigen. In someembodiments, the mutant or modified version of the firstlineage-specific cell-surface antigen escapes targeting by a cytotoxicagent that targets the corresponding wild-type lineage-specificcell-surface antigen. In some embodiments, the mutant or modifiedversion of the first lineage-specific cell-surface antigen retains itsbiological activity. In some embodiments, the mutant or modified versionof the first lineage-specific cell-surface antigen escapes targeting bya cytotoxic agent that targets the corresponding wild-typelineage-specific cell-surface antigen and retains its biologicalactivity. In some embodiments, the second gene has been modified oredited such that expression of the second lineage-specific cell-surfaceantigen is reduced or eliminated in the genetically engineeredhematopoietic cell (e.g., as compared with expression of thecorresponding endogenous or wild-type lineage-specific cell-surfaceantigen). In some embodiments, the second gene has been modified oredited such that the genetically engineered hematopoietic cell expressesa mutant or modified version of the second lineage-specific cell-surfaceantigen. In some embodiments, the mutant or modified version of thesecond lineage-specific cell-surface antigen escapes targeting by acytotoxic agent that targets the corresponding wild-typelineage-specific cell-surface antigen. In some embodiments, the mutantor modified version of the second lineage-specific cell-surface antigenretains its biological activity. In some embodiments, the mutant ormodified version of the second lineage-specific cell-surface antigenescapes targeting by a cytotoxic agent that targets the correspondingwild-type lineage-specific cell-surface antigen and retains itsbiological activity.

Another aspect of the present disclosure features a population ofgenetically engineered hematopoietic cells, wherein the geneticallyengineered hematopoietic cells of the population further comprise: (iii)a third gene encoding a third lineage-specific cell-surface antigen,which gene has been modified or edited. In some embodiments, thegenetically engineered hematopoietic cells of the population furthercomprise: (iv) a fourth gene encoding a fourth lineage-specificcell-surface antigen, which gene has been modified or edited. In someembodiments, the genetically engineered hematopoietic cells of thepopulation further comprise (v) a fifth gene encoding a fifthlineage-specific cell-surface antigen, which gene has been modified oredited. In some embodiments, any one or more of the gene(s) encoding alineage-specific cell-surface antigen has been modified or edited suchthat expression of the respective lineage-specific cell-surfaceantigen(s) is reduced or eliminated in the genetically engineeredhematopoietic cell (e.g., as compared with expression of thecorresponding endogenous or wild-type lineage-specific cell-surfaceantigen). In some embodiments, any one or more of the gene(s) encoding alineage-specific cell-surface antigen has been modified or edited suchthat the genetically engineered hematopoietic cell expresses a mutant ormodified version of the respective lineage-specific cell-surfaceantigen(s). In some embodiments, any one or more of the mutantlineage-specific cell-surface antigen(s) escapes targeting by acytotoxic agent that targets the corresponding wild-typelineage-specific cell-surface antigen. In some embodiments, any one ormore of the mutant or modified version lineage-specific cell-surfaceantigen(s) retains its biological activity. In some embodiments, any oneor more of the mutant lineage-specific cell-surface antigen(s) escapestargeting by a cytotoxic agent that targets the corresponding wild-typelineage-specific cell-surface antigen and retains its biologicalactivity. One aspect of the present disclosure features a population ofgenetically engineered hematopoietic cells, comprising: (i) a firstgroup of genetically engineered hematopoietic cells, which have geneticmodification or editing in a first gene encoding a firstlineage-specific cell-surface antigen, wherein the first group ofgenetically engineered hematopoietic cells (a) have reduced oreliminated expression of the first lineage-specific cell-surface antigenor (b) express a mutant of the first lineage-specific cell-surfaceantigen; and (ii) a second group of genetically engineered hematopoieticcells, which have genetic modification or editing in a second geneencoding a second lineage-specific cell-surface antigen, wherein thesecond group of genetically engineered hematopoietic cells (a) havereduced or eliminated expression of the second lineage-specificcell-surface antigen or (b) express a mutant of the secondlineage-specific cell-surface antigen. In some embodiments, the firstgroup of genetically engineered hematopoietic cells may overlap with thesecond group of genetically engineered hematopoietic cells, completelyor partially.

In some embodiments, the present disclosure provides a geneticallyengineered hematopoietic cell, comprising: (i) a gene encoding a CD19,which gene has been modified or edited and (ii) a gene encoding a CD33,which gene has been modified or edited. In some embodiments, the CD19gene has been modified or edited such that expression of the CD19antigen is reduced or eliminated in the genetically engineeredhematopoietic cell (e.g., as compared with expression of thecorresponding endogenous or wild-type CD19 antigen). In someembodiments, the CD19 gene has been modified or edited such that thegenetically engineered hematopoietic cell expresses a mutant or modifiedversion of the CD19 antigen. In some embodiments, the mutant or modifiedversion of the CD19 antigen escapes targeting by a cytotoxic agent thattargets the corresponding wild-type CD19 antigen. In some embodiments,the mutant or modified version of the CD19 antigen retains itsbiological activity. In some embodiments, the mutant or modified versionof the CD19 antigen escapes targeting by a cytotoxic agent that targetsthe corresponding wild-type CD19 antigen and retains its biologicalactivity. In some embodiments, the CD33 gene has been modified or editedsuch that expression of the CD33 antigen is reduced or eliminated in thegenetically engineered hematopoietic cell (e.g., as compared withexpression of the corresponding endogenous or wild-type CD33 antigen).In some embodiments, the CD33 gene has been modified or edited such thatthe genetically engineered hematopoietic cell expresses a mutant ormodified version of the CD33 antigen. In some embodiments, the mutant ormodified version of the CD33 antigen escapes targeting by a cytotoxicagent that targets the corresponding wild-type CD33 antigen. In someembodiments, the mutant or modified version of the CD33 antigen retainsits biological activity. In some embodiments, the mutant or modifiedversion of the CD33 antigen escapes targeting by a cytotoxic agent thattargets the corresponding wild-type CD33 antigen and retains itsbiological activity. In some embodiments of the genetically engineeredhematopoietic cell comprising: (i) a modified or edited gene encoding aCD19 and (ii) a modified or edited gene encoding a CD33, the geneencoding CD19 has been modified or edited such that the entire exon 2 isdeleted or a portion of exon 2 is deleted. In some embodiments of thegenetically engineered hematopoietic cell comprising: (i) a modified oredited gene encoding a CD19 and (ii) a modified or edited gene encodinga CD33, the gene encoding CD33 has been modified or edited such that theentire exon 2 is deleted or a portion of exon 2 is deleted. In someembodiments of the genetically engineered hematopoietic cell comprising:(i) a modified or edited gene encoding a CD19 and (ii) a modified oredited gene encoding a CD33, the gene encoding CD19 has been modified oredited such that the gene is truncated, has inserted and/or deletedsequences (e.g., resulting in scrambled, frameshift, or nonsensesequence), or the entire gene is deleted (e.g., effectively a knock-outgene). In some embodiments of the genetically engineered hematopoieticcell comprising: (i) a modified or edited gene encoding a CD19 and (ii)a modified or edited gene encoding a CD33, the gene encoding CD33 hasbeen modified or edited such that the gene is truncated, has insertedand/or deleted sequences (e.g., resulting in scrambled, frameshift, ornonsense sequence), or the entire gene is deleted (e.g., effectively aknock-out gene). In some embodiments of the genetically engineeredhematopoietic cell comprising: (i) a modified or edited gene encoding aCD19 and (ii) a modified or edited gene encoding a CD33, the geneencoding CD19 has been modified or edited such that the entire exon 2 ofCD19 is deleted or a portion of exon 2 od CD19 is deleted and the geneencoding CD33 has been modified or edited such that the CD33 gene istruncated, has inserted and/or deleted sequences (e.g., resulting inscrambled, frameshift, or nonsense sequence), or the entire CD33 gene isdeleted. In some embodiments of the genetically engineered hematopoieticcell comprising: (i) a modified or edited gene encoding a CD19 and (ii)a modified or edited gene encoding a CD33, the gene encoding CD33 hasbeen modified or edited such that the entire exon 2 of CD33 is deletedor a portion of exon 2 of CD33 is deleted and the gene encoding CD19 hasbeen modified or edited such that the CD19 gene is truncated, hasinserted and/or deleted sequences (e.g., resulting in scrambled,frameshift, or nonsense sequence), or the entire gene is deleted.

In some embodiments, the present disclosure provides a geneticallyengineered hematopoietic cell, comprising: (i) a gene encoding a CD19,which gene has been modified or edited such that the entire exon 2 isdeleted or a portion of exon 2 is deleted and (ii) a gene encoding aCD33, which gene has been modified or edited such that the entire exon 2is deleted or a portion of exon 2 is deleted. In some embodiments, themutant of the CD19 antigen with exon 2 deleted or a portion of exon 2deleted retains its biological activity. In some embodiments, the mutantof the CD19 antigen with exon 2 deleted or a portion of exon 2 deletedescapes targeting by a cytotoxic agent that targets the correspondingwild-type CD19 antigen and retains its biological activity. In someembodiments, the mutant of the CD33 antigen with exon 2 deleted or aportion of exon 2 deleted retains its biological activity. In someembodiments, the mutant of the CD33 antigen with exon 2 deleted or aportion of exon 2 deleted escapes targeting by a cytotoxic agent thattargets the corresponding wild-type CD33 antigen and retains itsbiological activity. In some embodiments, the present disclosureprovides a genetically engineered hematopoietic cell, comprising: (i) agene encoding a CD19, which gene has been modified or edited such thatthe entire exon 2 is deleted or a portion of exon 2 is deleted and (ii)a gene encoding a CD33, which gene has been modified or edited such thatthe entire exon 2 is deleted or a portion of exon 2 is deleted. In someembodiments, the present disclosure provides a genetically engineeredhematopoietic cell, comprising a gene encoding a CD19, which gene hasbeen modified or edited such that intron 1 and/or intron 2 in CD19 hasbeen modified or edited. In some embodiments, the genetically engineeredhematopoietic cell has a sequence deletion in intron 1 and/or intron 2of CD19 gene, e.g., either a portion of intron 1 and/or intron 2 of CD19is deleted or the entire intron 1 and/or intron 2 of CD19 is deleted. Insome embodiments, the present disclosure provides a geneticallyengineered hematopoietic cell, comprising a gene encoding a CD33, whichgene has been modified or edited such that intron 1 and/or intron 2 inCD33 has been modified or edited. In some embodiments, the geneticallyengineered hematopoietic cell has a sequence deletion in intron 1 and/orintron 2 of CD33 gene, e.g., either a portion of intron 1 and/or intron2 of CD33 is deleted or the entire intron 1 and/or intron 2 of CD33 isdeleted. In some embodiments, the present disclosure provides agenetically engineered hematopoietic cell, comprising: (i) a geneencoding a CD19, which gene has been modified or edited such that intron1 and/or intron 2 in CD19 has been modified or edited and (ii) a geneencoding a CD33, which gene has been modified or edited such that intron1 and/or intron 2 in CD33 has been modified or edited. In someembodiments, the mutant of the CD19 antigen with intron 1 and/or intron2 deleted or a portion of intron 1 and/or intron 2 deleted retains itsbiological activity. In some embodiments, the mutant of the CD19 antigenwith intron 1 and/or intron 2 deleted or a portion of intron 1 and/orintron 2 deleted escapes targeting by a cytotoxic agent that targets thecorresponding wild-type CD19 antigen and retains its biologicalactivity. In some embodiments, the mutant of the CD33 antigen withintron 1 and/or intron 2 deleted or a portion of intron 1 and/or intron2 deleted retains its biological activity. In some embodiments, themutant of the CD33 antigen with intron 1 and/or intron 2 deleted or aportion of intron 1 and/or intron 2 deleted escapes targeting by acytotoxic agent that targets the corresponding wild-type CD33 antigenand retains its biological activity.

Any of the genetically engineered hematopoietic cells or populations ofgenetically engineered hematopoietic cells described herein may behematopoietic stem cells (HSCs). In some instances, the HSCs areCD34+/CD33− cells. Any of the hematopoietic cells described herein canbe from bone marrow cells, cord blood cells, or peripheral bloodmononuclear cells (PBMCs). In some embodiments, the geneticallyengineered hematopoietic cell is a human hematopoietic cell. In someembodiments, any of the hematopoietic cells described herein are bonemarrow cells, cord blood cells, or peripheral blood mononuclear cells(PBMCs) derived from a human.

In some embodiments, the mutant of the first lineage-specificcell-surface antigen and/or the mutant of the second lineage-specificcell-surface antigen (and/or the mutant of a third, and/or fourth,and/or fifth lineage-specific cell-surface antigen) includes a mutatedor deleted non-essential epitope. Such a non-essential epitope in thefirst lineage-specific cell surface antigen and/or the non-essentialepitope in the second lineage-specific cell surface antigen (and/or thenon-essential epitope in the third, and/or fourth, and/or fifthlineage-specific cell-surface antigen) has at least 3 amino acids. Insome examples, the non-essential epitope in the first lineage-specificcell surface antigen and/or the non-essential epitope in the secondlineage-specific cell surface antigen (and/or the non-essential epitopein the third, and/or fourth, and/or fifth lineage-specific cell-surfaceantigen) is 6-10 amino acids. In some examples, the non-essentialepitope in the first lineage-specific cell surface antigen and/or thenon-essential epitope in the second lineage-specific cell surfaceantigen (and/or the non-essential epitope in the third, and/or fourth,and/or fifth lineage-specific cell-surface antigen) is 6-200 aminoacids. In some examples, the non-essential epitope in the firstlineage-specific cell surface antigen and/or the non-essential epitopein the second lineage-specific cell surface antigen (and/or thenon-essential epitope in the third, and/or fourth, and/or fifthlineage-specific cell-surface antigen) is 6-175 amino acids. In someexamples, the non-essential epitope in the first lineage-specific cellsurface antigen and/or the non-essential epitope in the secondlineage-specific cell surface antigen (and/or the non-essential epitopein the third, and/or fourth, and/or fifth lineage-specific cell-surfaceantigen) is 6-150 amino acids. In some examples, the non-essentialepitope in the first lineage-specific cell surface antigen and/or thenon-essential epitope in the second lineage-specific cell surfaceantigen (and/or the non-essential epitope in the third, and/or fourth,and/or fifth lineage-specific cell-surface antigen) is 6-125 aminoacids. In some examples, the non-essential epitope in the firstlineage-specific cell surface antigen and/or the non-essential epitopein the second lineage-specific cell surface antigen (and/or thenon-essential epitope in the third, and/or fourth, and/or fifthlineage-specific cell-surface antigen) is 6-100 amino acids. In someexamples, the non-essential epitope in the first lineage-specific cellsurface antigen and/or the non-essential epitope in the secondlineage-specific cell surface antigen (and/or the non-essential epitopein the third, and/or fourth, and/or fifth lineage-specific cell-surfaceantigen) is 6-75 amino acids. In some examples, the non-essentialepitope in the first lineage-specific cell surface antigen and/or thenon-essential epitope in the second lineage-specific cell surfaceantigen (and/or the non-essential epitope in the third, and/or fourth,and/or fifth lineage-specific cell-surface antigen) is 6-50 amino acids.In some examples, the non-essential epitope in the firstlineage-specific cell surface antigen and/or the non-essential epitopein the second lineage-specific cell surface antigen (and/or thenon-essential epitope in the third, and/or fourth, and/or fifthlineage-specific cell-surface antigen) is 6-25 amino acids. In someexamples, the non-essential epitope in the first lineage-specific cellsurface antigen and/or the non-essential epitope in the secondlineage-specific cell surface antigen (and/or the non-essential epitopein the third, and/or fourth, and/or fifth lineage-specific cell-surfaceantigen) is an entire exon or a portion of an exon.

In some embodiments at least one of the first and secondlineage-specific cell-surface antigens is associated with ahematopoietic malignancy. In some embodiments at least one of any of themodified or mutant lineage-specific cell-surface antigens is associatedwith a hematopoietic malignancy. Non-limiting examples include CD7,CD13, CD19, CD22, CD25, CD32, CD33, CD38, CD44, CD47, CD56, 96, CD117,CD123, CD135, CD174, CLL-1, folate receptor b, IL1RAP, MUC1,NKG2D/NKG2DL, TIM-3, and WT1. In some examples, the first and secondlineage-specific cell-surface antigens are selected from (a) CD19+CD33,(b) CD19+CD13, (c) CD19+CD123, (d) CD33+CD13, (e) CD33+CD123, (f)CD13+CD123.

In some embodiments, the modified lineage-specific cell-surface antigenis a type 1 lineage-specific cell-surface antigen. In some embodiments,at least one of the lineage-specific cell-surface antigens is a type 1lineage-specific cell-surface antigen. In some embodiments, thelineage-specific cell-surface antigen is CD19. In some embodiments, atleast one of the lineage-specific cell-surface antigens is CD19. In someembodiments, at least one of the first and second lineage-specificcell-surface antigens is a type 1 lineage-specific cell-surface antigen,for example, CD19. In some embodiments at least one of any of themodified or mutant lineage-specific cell-surface antigens is a type 1lineage-specific cell-surface antigen, for example, CD19. In someembodiments, both of the first and second lineage-specific cell-surfaceantigens are type 1 lineage-specific cell-surface antigens. In someembodiments, the genetic modification or editing of a CD19 gene (e.g.,an endogenous CD19 gene) occurs in an exon of the CD19 gene. In someembodiments, the genetic modification or editing of a CD19 gene (e.g.,an endogenous CD19 gene) occurs in exon 2 of the CD19 gene. In someembodiments, the genetic modification or editing of a CD19 gene (e.g.,an endogenous CD19 gene) occurs in one or more introns of the CD19 gene,e.g., including modification or editing of one or more introns thatresult in modification(s) in exon 2 of CD19. In some embodiments, thegenetic modification or editing of a CD19 gene (e.g., an endogenous CD19gene) results in mutation or deletion of exon 2 of a CD19 gene. In someembodiments, the genetic modification or editing of a CD19 gene (e.g.,an endogenous CD19 gene) results in deletion of the entire exon 2 of aCD19 gene or deletion of a portion of exon 2 of a CD19 gene. In someinstances, the mutated CD19 comprises the amino acid sequence of SEQ IDNO: 52.

In some embodiments, the genetic modification or editing of a CD19 gene(e.g., an endogenous CD19 gene) occurs in one or more introns of theCD19 gene, e.g., including modification or editing of one or moreintrons that result in modification(s) in exon 4 of CD19. In someembodiments, the genetic modification or editing of a CD19 gene (e.g.,an endogenous CD19 gene) results in mutation or deletion of exon 4 of aCD19 gene. In some embodiments, the genetic modification or editing of aCD19 gene (e.g., an endogenous CD19 gene) results in deletion of theentire exon 4 of a CD19 gene or deletion of a portion of exon 2 of aCD19 gene. In some instances, the mutated CD19 comprises the amino acidsequence of SEQ ID NO: 73.

In some embodiments, the modified lineage-specific cell-surface antigenis a type 2 lineage-specific cell-surface antigen. In some embodiments,at least one of the lineage-specific cell-surface antigens is a type 2lineage-specific cell-surface antigen. In some embodiments, thelineage-specific cell-surface antigen is CD33. In some embodiments, atleast one of the lineage-specific cell-surface antigens is CD33. In someembodiments, at least one of the first and second lineage-specificcell-surface antigens is a type 2 lineage-specific cell-surface antigen,for example, CD33. In some embodiments at least one of any of themodified or mutant lineage-specific cell-surface antigens is a type 2lineage-specific cell-surface antigen, for example, CD33. In someembodiments, both of the first and second lineage-specific cell-surfaceantigens are type 2 lineage-specific cell-surface antigens. In someembodiments, the genetic modification or editing of a CD33 gene (e.g.,an endogenous CD33 gene) occurs in an exon of the CD33 gene. In someembodiments, the genetic modification or editing of a CD33 gene (e.g.,an endogenous CD33 gene) occurs in exon 2 of the CD33 gene. In someembodiments, the genetic modification or editing of a CD33 gene (e.g.,an endogenous CD33 gene) occurs in one or more introns of the CD33 gene,e.g., including modification or editing of one or more introns thatresult in modification(s) in exon 2 of CD33. In some embodiments, thegenetic modification or editing of a CD33 gene (e.g., an endogenous CD33gene) occurs in intron 1 and intron 2 of the CD33 gene. In someembodiments, the genetic modification or editing of a CD33 gene (e.g.,an endogenous CD33 gene) results in mutation or deletion of exon 2 ofthe CD33 gene. In some embodiments, the genetic modification or editingof a CD33 gene (e.g., an endogenous CD33 gene) results in deletion ofthe entire exon 2 of the CD33 gene or deletion of a portion of exon 2 ofthe CD33 gene. In some embodiments, the genetic modification or editingof a CD33 gene (e.g., an endogenous CD33 gene) occurs in exon 3 of theCD33 gene. In some embodiments, the genetic modification or editing of aCD33 gene (e.g., an endogenous CD33 gene) occurs in one or more intronsof the CD33 gene, e.g., including modification or editing of one or moreintrons that result in modification(s) in exon 3 of CD33. In someembodiments, the genetic modification or editing of a CD33 gene (e.g.,an endogenous CD33 gene) results in mutation or deletion of exon 3 ofthe CD33 gene. In some embodiments, the genetic modification or editingof a CD33 gene (e.g., an endogenous CD33 gene) results in deletion ofthe entire exon 3 of the CD33 gene or deletion of a portion of exon 3 ofthe CD33 gene. In some examples, the second group of geneticallyengineered hematopoietic cells may contain genetic editing in exon 2 orexon 3 of a CD33 gene (e.g., including genetic modifications at one ormore introns that result in modifications in exon 2 or exon 3). In someexamples, the CD33 gene is an endogenous CD33 gene. Example CD33 mutantsinclude SEQ ID NO: 56 or SEQ ID NO: 58.

In some embodiments, the second lineage-specific cell surface antigen isa type 0 protein. In some embodiments, at least one of the first andsecond lineage-specific cell surface antigens is a type 0 protein. Insome embodiments at least one of any of the modified or mutantlineage-specific cell-surface antigens is a type 0 lineage-specificcell-surface antigen. In some embodiments, both of the first and secondlineage-specific cell-surface antigens are type 0 lineage-specificcell-surface antigens. In some embodiments, the genetic modification orediting of a Type 0 antigen gene occurs in an exon of the Type 0 gene.In some embodiments, the genetic modification or editing of a Type 0antigen occurs in exon 2 of the Type 0 antigen gene. In someembodiments, the genetic modification or editing of a Type 0 antigengene occurs in one or more introns of the Type 0 antigen gene, e.g.,including modification or editing of one or more introns that result inmodification(s) in exon 2 of the Type 0 antigen gene. In someembodiments, the genetic modification or editing of a Type 0 antigengene occurs in intron 1 and intron 2 of the Type 0 antigen gene.

Any of the genetically engineered hematopoietic cells described hereincan be produced by genomic editing. In some embodiments, the genomicediting does not involve an exogenous nuclease. In some embodiments, thegenomic editing involves adeno-associated virus vector mediatedhomologous recombination. In some embodiments, the genomic editinginvolves an exogenous nuclease. Exemplary approaches include the methodthat involve the use of a zinc finger nuclease (ZFN), a transcriptionactivator-like effector-based nuclease (TALEN), or a CRISPR-Cas system.In some embodiments, the CRISPR-Cas system comprises a Cas endonuclease.In some embodiments, the Cas endonuclease is a Cas9 endonuclease.

In another aspect, provided herein are methods for producing any of thegenetically engineered hematopoietic cells or populations of geneticallyengineered hematopoietic cells. In one aspect, provided herein is amethod for producing a population of genetically engineeredhematopoietic cells, the method comprising: (i) providing a populationof hematopoietic cells, and (ii) genetically modifying or editing atleast one lineage-specific cell surface antigen. In some embodiments,the at least one lineage-specific cell surface antigen is geneticallymodified via CRISPR to produce the population of genetically engineeredhematopoietic cells. In one aspect, provided herein is a method forproducing a population of genetically engineered hematopoietic cells,the method comprising: (i) providing a population of hematopoieticcells, and (ii) genetically modifying or editing a firstlineage-specific cell surface antigen, or genetically modifying orediting a second lineage-specific cell surface antigen, or geneticallymodifying or editing a first lineage-specific cell surface antigen and asecond lineage-specific cell surface antigen in the population ofhematopoietic cells via CRISPR to produce the population of geneticallyengineered hematopoietic cells. In one aspect, provided herein is amethod for producing a population of genetically engineeredhematopoietic cells, the method comprising: (i) providing a populationof hematopoietic cells, and (ii) genetically modifying or editing afirst lineage-specific cell surface antigen and genetically modifying orediting a second lineage-specific cell surface antigen in the populationof hematopoietic cells to produce the population of geneticallyengineered hematopoietic cells. In some embodiments, the method forproducing a population of genetically engineered hematopoietic cellsfurther comprises (iii) genetically modifying or editing one or moreother lineage-specific cell surface antigen(s) in the population ofhematopoietic cells to produce the population of genetically engineeredhematopoietic cells. In any of these methods, the genetically engineeredhematopoietic cell is a human cell.

In one aspect, provided herein is a method for producing a geneticallyengineered hematopoietic cell, the method comprising: (i) providing ahematopoietic cell, and (ii) genetically modifying or editing a firstlineage-specific cell surface antigen, or genetically modifying orediting a second lineage-specific cell surface antigen, or geneticallymodifying or editing a first lineage-specific cell surface antigen and asecond lineage-specific cell surface antigen in the hematopoietic cellvia CRISPR to produce the genetically engineered hematopoietic cell. Inone aspect, provided herein is a method for producing a geneticallyengineered hematopoietic cell, the method comprising: (i) providing ahematopoietic cell, and (ii) genetically modifying or editing a firstlineage-specific cell surface antigen and genetically modifying orediting a second lineage-specific cell surface antigen to produce thegenetically engineered hematopoietic cell. In some embodiments, themethod for producing a genetically engineered hematopoietic cell furthercomprises (iii) genetically modifying or editing one or more otherlineage-specific cell surface antigen(s) to produce the geneticallyengineered hematopoietic cell. In any of these methods, the geneticallyengineered hematopoietic cell is a human cell.

In one aspect, provided herein is a method for producing a population ofgenetically engineered hematopoietic cells, the method comprising: (i)providing a population of hematopoietic cells, and (ii) geneticallymodifying or editing a CD19 gene, or genetically modifying or editing aCD33 gene, or genetically modifying or editing a CD19 gene and a CD33gene in the population of hematopoietic cells via CRISPR to produce thegenetically engineered hematopoietic cells. In one aspect, providedherein is a method for producing a population of genetically engineeredhematopoietic cells, the method comprising: (i) providing a populationof hematopoietic cells, and (ii) genetically modifying or editing a CD19gene and genetically modifying or editing a CD33 gene in the populationof hematopoietic cells to produce the population of geneticallyengineered hematopoietic cells. In one aspect, provided herein is amethod for producing a genetically engineered hematopoietic cell, themethod comprising: (i) providing a hematopoietic cell, and (ii)genetically modifying or editing a CD19 gene, or genetically modifyingor editing a CD33 gene, or genetically modifying or editing a CD19 geneand genetically modifying or editing a CD33 gene in the hematopoieticcell via CRISPR to produce the genetically engineered hematopoieticcell. In one aspect, provided herein is a method for producing agenetically engineered hematopoietic cell, the method comprising: (i)providing a hematopoietic cell, and (ii) genetically modifying orediting a CD19 gene and genetically modifying or editing a CD33 gene toproduce the genetically engineered hematopoietic cell. In someembodiments, the method for producing a genetically engineeredhematopoietic cell or population of genetically engineered hematopoieticcells further comprises (iii) genetically modifying or editing one ormore other lineage-specific cell surface antigen(s) to produce thegenetically engineered hematopoietic cell. In any of these methods, thegenetically engineered hematopoietic cell is a human cell.

In some embodiments, the genetic editing of the CD19 gene involves oneor more guide nucleic acid molecules that target one or more introns ofCD19. In some embodiments, the genetic editing of the CD33 gene involvesone or more guide nucleic acid molecules that target one or more intronsof CD33. In some embodiments, the genetic editing of the CD33 geneinvolves one or more guide nucleic acid molecules that do not target theCD33 pseudogene upstream of the CD33 gene. In some embodiments, thegenetic editing of the CD33 gene involves one or more guide nucleic acidmolecules that (a) target one or more introns of CD33 and (b) do nottarget the CD33 pseudogene upstream of the CD33 gene. In someembodiments, the introns of the CD19 gene comprise intron 1 and intron2. In some embodiments, the introns of the CD33 gene comprise intron 1and intron 2. In some embodiments, the genetic editing of CD19 resultsin exclusion of exon 2 of the CD19 gene. In some embodiments, thegenetic editing of CD19 results in exclusion of exon 4 of the CD19 gene.In some embodiments, the genetic editing of CD33 results in exclusion ofexon 2 of the CD33 gene. In some embodiments, the genetic editing of theCD33 gene involves one or more guide nucleic acid molecules that targetexon 3 of CD33. In some embodiments, the genetic editing of the CD33gene involves at least one guide nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 67.

Further, provided herein is a method for producing geneticallyengineered hematopoietic cells, the method comprising: (i) providing apopulation of hematopoietic cells, and (ii) genetically editing a CD19gene, a CD33 gene, or both a CD19 and a CD33 gene in the population ofhematopoietic cells via CRISPR to produce the genetically engineeredhematopoietic cells, wherein the genetic editing of the CD19 geneinvolves at least one guide nucleic acid molecule comprising thenucleotide sequence of SEQ ID NOs: 14-26, 67, and 69-72, and/or whereinthe genetic editing of the CD33 gene involves at least one guide nucleicacid molecule comprising the nucleotide sequence of SEQ ID NOs: 27-50and 68. In some embodiments, the method for producing geneticallyengineered hematopoietic cells comprises genetically editing a CD33 genein the population of hematopoietic cells via CRISPR to produce thegenetically engineered hematopoietic cells, wherein the genetic editingof the CD33 gene involves at least one guide nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 67.

In some embodiments, step (ii) is performed by genetic editing of both aCD19 gene and a CD33 gene in the population of hematopoietic cells viaCRISPR to produce the genetically engineered hematopoietic cells. Thegenetic editing of the CD19 gene involves a guide nucleic acidcomprising the nucleotide sequence of SEQ ID NO: 67, and/or the geneticediting of the CD33 gene involves a guide nucleic acid comprising thenucleotide sequence of SEQ ID NO: 68.

In any of the methods described herein, the hematopoietic cells can beHSCs, for example, CD34+/CD33− cells. The hematopoietic cells can befrom bone marrow cells, cord blood cells, or peripheral bloodmononuclear cells (PBMCs). In some embodiments, the hematopoietic cellsare from human bone marrow cells, human cord blood cells, or humanperipheral blood mononuclear cells (PBMCs). Also provided herein aregenetically engineered hematopoietic cells having one of more of thefollowing features:

-   -   (a) carry a genetically edited CD19 gene capable of expressing a        mutant CD19 comprising the amino acid sequence of SEQ ID NO: 52        or 73 and/or a genetically edited CD33 gene capable of        expressing a mutant CD33 comprising the amino acid sequence of        SEQ ID NO: 56 or SEQ ID NO: 58;    -   (b) carry a genetically edited CD19 gene capable of expressing a        mutant CD19 comprising the amino acid sequence of SEQ ID NO: 52        or 73 and a genetically edited CD33 gene capable of expressing a        mutant CD33 comprising the amino acid sequence of SEQ ID NO: 56;    -   (c) exon 2 of CD33 gene in the hematopoietic cell is modified        and wherein one or more portions of the CD33 pseudogene are not        modified;    -   (d) exon 2 of CD33 gene in the hematopoietic cell is deleted and        wherein one or more portions of the CD33 pseudogene are not        modified;    -   (e) exon 2 of CD33 gene in the hematopoietic cell is modified        and wherein one or more portions of the CD33 pseudogene are not        modified by deletion or mutation that causes a frameshift.    -   (f) exon 2 of CD33 gene in the hematopoietic cell is modified        and wherein the one or more portion(s) of the CD33 pseudogene        that is not modified by deletion or mutation that causes a        frameshift is selected from Exon 1, intron1, Exon 2, and        combinations thereof;    -   (g) exon 2 of CD33 gene in the hematopoietic cell is modified        and wherein the one or more portion(s) of the CD33 pseudogene        that is not modified by deletion or mutation that causes a        frameshift is selected from sequence(s) in Exon 1, intron 1,        and/or Exon 2, that share sequence homology, respectively, with        sequence(s) in Exon 1, intron 1, and/or Exon 2 of CD33.

Further, genetically engineered hematopoietic cells produced by anymethod disclosed herein are also within the scope of the presentdisclosure.

Also provided herein is a population of genetically engineeredhematopoietic stem cells, wherein at least 50% (e.g., at least 60%, 70%,75%, 80%, 85%, 90%, or 95%) of the hematopoietic stem cells thereincarry both a genetically edited CD19 gene and a genetically edited CD33gene. In some instances, the genetically edited CD19 gene is capable ofexpressing a CD19 mutant comprising the amino acid sequence of SEQ IDNO: 52 or 73. Alternatively or in addition, the genetically edited CD33gene is capable of expressing a CD33 mutant comprising the amino acidsequence of SEQ ID NO: 56 or SEQ ID NO: 58.

Moreover, provided herein is a method of treating a hematopoieticmalignancy (e.g., AML), comprising administering to a subject in needthereof a population of genetically engineered hematopoietic cells asdisclosed herein. The method may further comprise administering to thesubject an effective amount of a first immunotherapeutic agent. In someinstances, the first immunotherapeutic agent is a cytotoxic agent thattargets cells expressing either the first lineage-specific cell-surfaceantigen or the second lineage-specific cell-surface antigen.

In some examples, the first immunotherapeutic agent is a cytotoxic agentthat targets cells expressing the first lineage-specific cell-surfaceantigen, and the method further comprises administering to the subject asecond immunotherapeutic agent when the hematopoietic malignancyrelapses in the subject. The second immunotherapeutic agent may be acytotoxic agent that targets cells expressing the secondlineage-specific cell-surface antigen. In one example, the firstimmunotherapeutic agent, the second immunotherapeutic agent, or both areCAR-T cells. In one example, the first immunotherapeutic agent, thesecond immunotherapeutic agent, or both are antibody drug conjugates.

The disclosure also provides methods of protecting hematopoietic stemcells from immunotherapy in a subject in need thereof, wherein thetherapy targets one or more lineage-specific antigen(s). In someembodiments, the methods comprise administering a modified hematopoieticstem cell to a subject, wherein the stem cell comprises one or moregene(s) encoding the lineage-specific antigen(s) being targeted by theimmunotherapy, and wherein the gene(s) are modified, mutated or edited.In some embodiments of the methods, the gene(s) are modified such thatexpression of the gene(s) results in modified, mutated protein(s) orcomplete knockout(s) of the protein or combinations thereof. In someembodiments of the method, modified, mutated protein(s) or completeknockout(s) prevent the immunotherapy from targeting the hematopoieticstem cells comprising the mutated gene(s). In some embodiments of themethods, the editing results in expression of one or morelineage-specific antigen(s), which contain a partial deletion. In someembodiments of the methods, the partial deletion compasses an entireexon or a portion of an exon. In some embodiments of the methods, theimmunotherapy administered includes one or more antibody-drugconjugate(s), which can be administered concurrently or sequentially. Insome embodiments of the methods, the immunotherapy administered includescells expressing one or more chimeric antigen receptors or a pool of 2or more cells, each expressing a different chimeric antigen receptor,which can be administered concurrently or sequentially. In oneembodiment, the disclosure provides a method of protecting hematopoieticstem cells from one or more chimeric antigen receptor T cell therapiestargeting one or more lineage specific antigen(s) in a subject in needthereof, wherein the hematopoietic stem cells are administered, andwherein the hematopoietic stem cells are modified such that expressionof the gene(s) encoding one or more lineage specific antigen(s) resultin modified, mutated forms or complete knockout(s) of the lineagespecific antigen(s) targeted by the CART(s). In one embodiment of thismethod, the mutated form(s) of the one or more lineage specificantigen(s) lack an exon, e.g., an exon which comprises the CART orantibody-drug conjugate epitope. In any of these methods, the modifiedhematopoietic cell can be any of the modified hematopoietic cellsdescribed here and elsewhere herein.

Such modified hematopoietic stems cells can be generated using geneediting technologies, e.g. CRISPR, as described herein. As describedelsewhere, CRISPR methodology can be used to delete a portion or anentire gene of interest. In some embodiments, CRISPR methodology can beused to delete one or more exons comprising a targeted epitope. In someinstances, it is beneficial to target one or more flanking intronsequences to excise an exon. In some instances, the exon sequence itselfmay be targeted by CRISPR, however, current conventional CRISPRtherapies may lead to small insertions and deletions, which can lead toframeshift and truncated non-functional proteins. To avoid unintentionalknockouts, the intron sequences may be beneficial to target so as tomore precisely edit the exon sequence of interest.

Accordingly, the disclosure also provides methods for generatingmodified hematopoietic stem cells, comprising introducing one or moreguide RNAs capable of editing one or more gene(s) encoding one or morelineage specific antigen(s) targeted by one or more chimeric antigenreceptor(s). In some embodiments of the methods, the editing results inexpression of lineage specific antigen(s) lacking an exon. In someembodiments, the editing results complete knockout of lineage specificantigen(s). In some embodiments of the methods, the editing results in acombination of expression of lineage specific antigen(s) lacking an exonand complete knockout of lineage specific antigen(s) in one cell or acell population. In some embodiments, the one or more guide RNAs areselected to target one or more introns. In some embodiments of themethod, targeting of adjacent introns results in excision of the genesequence encoding the exon between the two introns.

Targeting one intron may result in the generation of a new splice site,resulting in excision of the gene sequence encoding the adjacent exon.Exon skipping using a single guide RNA has been described (e.g., Mou etal., Genome Biology 201718:108). Accordingly, in some embodiments, oneintron may be targeted according to the methods described herein.

In some embodiments, a method of protecting hematopoietic stem cellsfrom immunotherapy may be used in a subject in need thereof, wherein thetherapy targets one or more lineage-specific antigen(s). In someembodiments, the methods comprise administering a modified hematopoieticstem cell to a subject, wherein the stem cell comprises one or moregene(s) encoding the lineage-specific antigen(s) CD19 and/or CD33 beingtargeted by the immunotherapy, and wherein the CD19 and/or CD33 gene(s)are modified, mutated or edited. In some embodiments of the methods, theCD19 and/or CD33 gene(s) are modified such that expression result inmodified, mutated CD19 and/or CD33 or complete knockout(s) of the CD19and/or CD33 or combinations thereof. In some embodiments of the method,modified, mutated CD19 and/or CD33 protein(s) or complete CD19 and/orCD33 knockout(s) prevent the immunotherapy from targeting thehematopoietic stem cells comprising the mutated gene(s). In someembodiments of the methods, the editing results in expression CD19and/or CD33, which contain a partial deletion. In some embodiments ofthe methods, the partial deletion compasses an entire exon or a portionof an exon, e.g., exon2 of CD33 and/or CD19. In some embodiments of themethods, the immunotherapy administered includes one or moreantibody-drug conjugate(s) directed against CD19 and/or CD33, which canbe administered concurrently or sequentially. In some embodiments of themethods, the immunotherapy administered includes cells expressing one ormore chimeric antigen receptors directed against CD19 and/or CD33 or apool of 2 cell populations, one expressing chimeric antigen receptordirected against CD19 and the other expressing a chimeric antigenreceptor directed against CD33. The cells expressing chimeric antigenreceptor directed against CD19 can be administered concurrently orsequentially with the cells expressing chimeric antigen receptordirected against CD33. In one embodiment, the disclosure provides amethod of protecting hematopoietic stem cells from one or more chimericantigen receptor T cell therapies targeting CD19 and/or CD33 in asubject in need thereof, wherein the hematopoietic stem cells areadministered, and wherein the hematopoietic stem cells are modified suchthat expression of the gene(s) encoding CD19 and/or CD33 result inmodified, mutated forms or complete knockout(s) of the CD19 and/or CD33.In one embodiment of this method, the mutated form(s) of CD19 and/orCD33 lack an exon, e.g., an exon which comprises the CART epitope.

Accordingly, the disclosure also provides methods for generatingmodified hematopoietic stem cells, comprising introducing one or moreguide RNAs capable of editing one or more gene(s) encoding CD19 and/orCD33, wherein CD19 and/or CD33 are targeted by one or more chimericantigen receptor(s) or antibody-drug conjugates. In some embodiments ofthe methods, the editing results in expression of CD19 and/or CD33lacking an exon. In some embodiments, the editing results completeknockout of CD19 and/or CD33. In some embodiments of the methods, theediting results in a combination of expression of lineage specificantigen(s) lacking an exon and complete knockout of CD19 and/or CD33 inone cell or a cell population. In some embodiments, the one or moreguide RNAs are selected to target one or more introns. In someembodiments of the method, targeting of adjacent introns results inexcision of the gene sequence encoding the exon between the two introns.

ENUMERATED EMBODIMENTS

1. A population of genetically engineered hematopoietic cells,comprising:

(i) a first group of genetically engineered hematopoietic cells, whichhave genetic editing in a first gene encoding a first lineage-specificcell-surface antigen, wherein the first group of genetically engineeredhematopoietic cells (a) have reduced or eliminated expression of thefirst lineage-specific cell-surface antigen or (b) express a mutant ofthe first lineage-specific cell-surface antigen; and

(ii) a second group of genetically engineered hematopoietic cells, whichhave genetic editing in a second gene encoding a second lineage-specificcell-surface antigen, wherein the second group of genetically engineeredhematopoietic cells (a) have reduced or eliminated expression of thesecond lineage-specific cell-surface antigen or (b) express a mutant ofthe second lineage-specific cell-surface antigen.

2. The population of genetically engineered hematopoietic cells ofembodiment 1, wherein the first group of genetically engineeredhematopoietic cells overlaps with the second group of geneticallyengineered hematopoietic cells.

3. A population of genetically engineered hematopoietic cells, whereinone or more cells of the population:

(i) have reduced or eliminated expression of a first lineage-specificcell-surface antigen relative to a wild-type counterpart cell, orexpress a mutant of the first lineage-specific cell-surface antigen; and

(ii) have reduced or eliminated expression of a second lineage-specificcell-surface antigen relative to a wild-type counterpart cell, orexpress a mutant of the second lineage-specific cell-surface antigen.

4. The population of embodiment 3, wherein the reduction in expressionof the first lineage-specific cell-surface antigen, second firstlineage-specific cell-surface antigen, or both, is to less than or equalto 50%, 40%, 30%, 20%, 10%, 5%, 2%, or 1% of the level in a wild-typecounterpart cell.

5. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein the first lineage-specificcell-surface antigen (e.g., CD19) is expressed in a primary cancer in asubject and the second lineage-specific cell-surface antigen (e.g.,CD33) is expressed in a relapsed cancer in the subject.

6. The population of genetically engineered hematopoietic cells ofembodiment 1 or 2, wherein the first lineage-specific cell-surfaceantigen (e.g., CD33) is expressed in a first sub-population of cancercells in a subject, and the second lineage-specific cell-surface antigen(e.g., CD123 or CLL-1) is expressed in a second sub-population of cancercells in the subject.

7. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein at least 40%, 50%, 60%, 70%, 75%,80%, 85%, 90%, or 95% of cells in the population have genetic editing(e.g., comprise an indel or comprise a deletion) at both allelesencoding the first lineage-specific cell-surface antigen.

8. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein at least 40%, 50%, 60%, 70%, 75%,80%, 85%, 90%, or 95% of cells in the population have genetic editing(e.g., comprise an indel or comprise a deletion) at both allelesencoding the second lineage-specific cell-surface antigen.

9. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein at least 40%, 50%, 60%, 70%, 75%,80%, 85%, 90%, or 95% of cells in the population have genetic editing(e.g., comprise an indel or comprise a deletion) at both allelesencoding the first lineage-specific cell-surface antigen and at bothalleles encoding the second lineage-specific cell-surface antigen.

10. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein at least 40%, 50%, 60%, 70%, 75%,80%, 85%, 90%, or 95% of copies of the first gene (encoding thelineage-specific cell-surface antigen) in the population of cells havegenetic editing, e.g., as measured using PCR, e.g., according to anassay of Example 1.

11. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein at least 40%, 50%, 60%, 70%, 75%,80%, 85%, 90%, or 95% of copies of the second gene (encoding thelineage-specific cell-surface antigen) in the population of cells havegenetic editing, e.g., as measured using PCR, e.g., according to anassay of Example 1.

12. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein at least 40%, 50%, 60%, 70%, 75%,80%, 85%, 90%, or 95% of copies of the first and second genes (encodingthe first and second lineage-specific cell-surface antigens,respectively) in the population of cells have genetic editing, e.g., asmeasured using PCR, e.g., according to an assay of Example 1.

13. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein at least 60%, 70%, 75%, 80%, 85%,90%, or 95% of cells in the population (or cells differentiated fromcells in the population) are negative for the first lineage-specificcell-surface antigen.

14. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein at least 60%, 70%, 75%, 80%, 85%,90%, or 95% of cells in the population (or cells differentiated fromcells in the population) are negative for the second lineage-specificcell-surface antigen.

15. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein at least 60%, 70%, 75%, 80%, 85%,90%, or 95% of cells in the population (or cells differentiated fromcells in the population) are negative for both of the firstlineage-specific cell-surface antigen and the second lineage-specificcell-surface antigen.

16. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein surface levels of the firstlineage-specific cell-surface antigen in the population (or cellsdifferentiated from cells in the population) are less than 50%, 40%,30%, 20%, 10%, 5%, 2%, or 1% of surface levels of the firstlineage-specific cell-surface antigen in wild-type counterpart cells.

17. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein surface levels of the secondlineage-specific cell-surface antigen in the population (or cellsdifferentiated from cells in the population) are less than 50%, 40%,30%, 20%, 10%, 5%, 2%, or 1% of surface levels of the secondlineage-specific cell-surface antigen in wild-type counterpart cells.

18. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein intracellular levels of the firstlineage-specific antigen in the population (or cells differentiated fromcells in the population) are less than 50%, 40%, 30%, 20%, 10%, 5%, 2%,or 1% of intracellular levels of the first lineage-specific cell-surfaceantigen in wild-type counterpart cells.

19. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein intracellular levels of the secondlineage-specific antigen in the population (or cells differentiated fromcells in the population) are less than 50%, 40%, 30%, 20%, 10%, 5%, 2%,or 1% of intracellular levels of the second lineage-specificcell-surface antigen in wild-type counterpart cells.

20. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein the first and second genes arechosen from Table 1A.

21. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein the first and secondlineage-specific cell-surface antigens are chosen from Table 1A.

22. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, which comprises a plurality of HSCs and/orHPCs.

23. The population of genetically engineered hematopoietic cells ofembodiment 22, which retains differentiation potential, e.g., in an invitro CFU assay, e.g., as described in Example 1 herein.

24. The population of genetically engineered hematopoietic cells ofembodiment 23, wherein the cells form at least 1, 2, 3, 4, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, or 200 total colonies per 250 cells ina CFU assay, e.g., an assay of Example 1 herein.

25. The population of genetically engineered hematopoietic cells ofembodiment 23 or 24, wherein the cells form at least 1, 2, 3, 4, 5, 10,or 20 CFU-GEMM colonies per 250 cells in a CFU assay, e.g., an assay ofExample 1 herein.

26. The population of genetically engineered hematopoietic cells of anyof embodiments 23-25, wherein the cells form at least 1, 2, 3, 4, 5, 10,20, 30, 40, 50, 60, 70, 80, 90, or 100 CFU-GM colonies per 250 cells ina CFU assay, e.g., an assay of Example 1 herein.

27. The population of genetically engineered hematopoietic cells of anyof embodiments 23-26, wherein the cells form at least 1, 2, 3, 4, 5, 10,20, 30, 40, 50, 60, 70, 80, 90, or 100 BFU-E colonies per 250 cells in aCFU assay, e.g., an assay of Example 1 herein.

28. The population of genetically engineered hematopoietic cells of anyof embodiments 23-27, wherein the number of BFU-E colonies is about30%-150%, 35-135%, 40-120%, or 50%-100% of the number of CFU-GM colonieswhen assayed in a CFU assay (e.g., an assay of Example 1 herein).

29. The population of genetically engineered hematopoietic cells of anyof embodiments 23-28, wherein the number of CFU-GEMM colonies is about1-15%, 1-10%, or 1.5-5.0% of the number of CFU-GM colonies when assayedin a CFU assay (e.g., an assay of Example 1 herein).

30. The population of genetically engineered hematopoietic cells of anyof embodiments 23-29, wherein the number of CFU-GEMM colonies is about1-30%, 2-20%, or 3-10% of the number of BFU-E colonies when assayed in aCFU assay (e.g., an assay of Example 1 herein).

31. The population of genetically engineered hematopoietic cells of anyof embodiments 23-30, wherein one, two, three, or all of:

a) the number of BFU-E colonies formed by the cells in a CFU assay iswithin about 5%, 10%, 20%, or 30% of the number of BFU-E colonies formedby otherwise similar, unmodified cells;

b) the number of CFU-GM colonies formed by the cells in a CFU assay iswithin about 5%, 10%, 20%, or 30% of the number of CFU-GM coloniesformed by otherwise similar, unmodified cells;

c) the number of CFU-GEMM colonies formed by the cells in a CFU assay iswithin about 5%, 10%, 20%, or 30% of the number of CFU-GEMM coloniesformed by otherwise similar, unmodified cells; and

d) the total number of colonies formed by the cells in a CFU assay iswithin about 5%, 10%, 20%, or 30% of the total number of colonies formedby otherwise similar, unmodified cells.

32. The population of genetically engineered hematopoietic cells of anyof embodiments 23-31, wherein the cells can give rise to differentiatedmyeloid cells.

33. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, which are capable of growing in culture,e.g., of increasing by at least 2, 3, 4, 5, or 10-fold (e.g., over 8days, e.g., in conditions according to Example 1).

34. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, which have a viability of at least 50%,60%, 70%, 75%, 80%, 85%, or 90% (e.g., after 2, 4, 6, 8, or 10 days),e.g., in conditions according to Example 1 or Example 4.

35. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, which are capable of engraftment, e.g., toproduce at least 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, or 10% of CD45+ cells inperipheral blood of a subject, e.g., according to an assay of Example 1.

36. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, which can produce at least 0.1%, 0.2%,0.5%, 1%, 2%, 5%, 10%, 20%, 40%, 60%, or 80% of B cells in peripheralblood of a subject, e.g., according to an assay of Example 1.

37. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, which are resistant to a firstimmunotherapeutic agent that targets the first lineage-specificcell-surface antigen, e.g., wherein the IC₅₀ the first immunotherapeuticagent for the population of cells is greater than the IC₅₀ of the firstimmunotherapeutic agent for control cells (e.g., wherein the controlcells are wild-type counterpart cells), e.g., by at least 2, 3, 4, 5,10, 20, 50, or 100-fold, e.g., in an assay of Example 2.

38. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, which are resistant to a secondimmunotherapeutic agent that targets the second lineage-specificcell-surface antigen, e.g., wherein the IC₅₀ the secondimmunotherapeutic agent for the population of cells is greater than theIC₅₀ of the second immunotherapeutic agent for control cells (e.g.,wherein the control cells are wild-type counterpart cells), e.g., by atleast 2, 3, 4, 5, 10, 20, 50, or 100-fold, e.g., in an assay of Example2.

39. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, which are resistant to a firstimmunotherapeutic agent that targets the first lineage-specificcell-surface antigen and a second immunotherapeutic agent that targetsthe second lineage-specific cell-surface antigen, e.g., wherein thecells show a specific killing of less than 50%, 40%, 35%, 30%, 25%, 20%,or 15%, e.g., in an in vitro cytotoxicity assay, e.g., in an assay ofExample 9.

40. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein about 5-10%, 10-20%, 20-30%,30-40%, 40-50%, 50-60%, 60-70%, 70-80%, or 80-90% of cells in thepopulation substantially lack cell surface expression of both of thefirst lineage-specific cell-surface antigen and the secondlineage-specific cell-surface antigen.

41. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein about 5-10%, 10-20%, 20-30%,30-40%, 40-50%, 50-60%, 60-70%, 70-80%, or 80-90% of cells in thepopulation comprise a mutation of at least one allele of the firstlineage-specific cell-surface antigen and a mutation of at least oneallele of the second lineage-specific cell-surface antigen.

42. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein about 5-10%, 10-20%, 20-30%,30-40%, 40-50%, 50-60%, 60-70%, 70-80%, or 80-90% of cells in thepopulation comprise mutations at two alleles of the firstlineage-specific cell-surface antigen and mutations at two alleles ofthe second lineage-specific cell-surface antigen.

43. The population of genetically engineered hematopoietic cells of anyof embodiments 1-42, wherein:

(a) the first lineage-specific cell-surface antigen is CD33 and thesecond lineage-specific cell-surface antigen is CD123;

(b) the population of cells comprises HSCs; and

(c) at least 20%, 30%, 40%, 50%, or 60% of cells in the populationsubstantially lack cell surface expression of both of CD123 and CD33.

44. The population of genetically engineered hematopoietic cells of anyof embodiments 1-42, wherein:

(a) the first lineage-specific cell-surface antigen is CD33 and thesecond lineage-specific cell-surface antigen is CLL-1;

(b) the population of cells comprises HSCs; and

(c) at least 20%, 30%, 40%, 50%, or 60% of cells in the populationsubstantially lack cell surface expression of both of CLL1 and CD33.

45. The population of genetically engineered hematopoietic cells of anyof embodiments 1-42, wherein:

(a) the first lineage-specific cell-surface antigen is CD123 and thesecond lineage-specific cell-surface antigen is CLL-1;

(b) the population of cells comprises HSCs; and

(c) at least 20%, 30%, 40%, 50%, or 60% of cells in the populationsubstantially lack cell surface expression of both of CLL1 and CD123.

46. The population of genetically engineered hematopoietic cells of anyof embodiments 1-42, wherein:

(a) the first lineage-specific cell-surface antigen is CD19 and thesecond lineage-specific cell-surface antigen is CD33;

(b) the population of cells comprises HSCs; and

(c) at least 20%, 30%, 40%, 50%, or 60% of cells in the populationsubstantially lack cell surface expression of both of CD19 and CD33.

47. The population of any of embodiments 43-46, wherein (d) the geneticediting of the first gene comprises a frameshift mutation and thegenetic editing of the second gene comprises a frameshift mutation.

48. The population of genetically engineered hematopoietic cells of anyof the preceding embodiments, wherein the hematopoietic cells arehematopoietic stem cells (HSCs).

49. The population of genetically engineered hematopoietic cells ofembodiment 48, wherein the HSCs are CD34+/CD33− cells.

50. The population of genetically engineered hematopoietic cells of anyone of embodiments 1-49, which the hematopoietic cells are from bonemarrow cells, cord blood cells, or peripheral blood mononuclear cells(PBMCs).

51. The population of genetically engineered hematopoietic cells of anyone of embodiments 1-40, wherein the mutant of the firstlineage-specific cell-surface antigen and/or the mutant of the secondlineage-specific cell-surface antigen includes a mutated non-essentialepitope.

52. The population of genetically engineered hematopoietic cells of anyof embodiments 1-51, wherein one or both of: the genetic editing of thegene encoding the first lineage-specific cell surface antigen comprisesa frameshift mutation, and the genetic editing of the second genecomprises a frameshift mutation.

53. The population of genetically engineered hematopoietic cells ofembodiment 52, wherein the frameshift mutation comprises an insertion ordeletion of less than 20, 15, 10, 5, 4, 3, or 2 nucleotides.

54. The population genetically engineered hematopoietic cells of any ofthe preceding embodiments, wherein the genetic editing comprises genomeediting.

55. The population of genetically engineered hematopoietic cells of anyof embodiments 1-54, wherein one or more cells in the population arenegative for one or both of the first lineage-specific cell-surfaceantigen and the second first lineage-specific cell-surface antigen.

56. The population of genetically engineered hematopoietic cells of anyof embodiments 1-55, wherein the CD33 pseudogene is not modified in oneor more (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, orall) of the cells of the population.

57. The population of genetically engineered hematopoietic cells of anyof embodiments 1-56, wherein the average number of off-target geneticedits in the cell population is less than 3, 2, or 1 per cell.

58. The population of genetically engineered hematopoietic cells ofembodiment 57, wherein the non-essential epitope in the firstlineage-specific cell surface antigen and/or the non-essential epitopein the second lineage-specific cell surface antigen has at least 3 aminoacids.

59. The population of genetically engineered hematopoietic cells ofembodiment 58, wherein the non-essential epitope in the firstlineage-specific cell surface antigen and/or the non-essential epitopein the second lineage-specific cell surface antigen is 6-10 amino acids.

60. The population of genetically engineered hematopoietic cells of anyone of embodiments 1-59, wherein at least one of the first and secondlineage-specific cell-surface antigens is a type 1 lineage-specificcell-surface antigen.

61. The population of genetically engineered hematopoietic cells ofembodiment 60, wherein the type 1 lineage-specific cell-surface antigenis CD19.

62. The population of genetically engineered hematopoietic cells ofembodiment 61, wherein the first group of genetically engineeredhematopoietic cells contain genetic editing in exon 2 or exon 4 of aCD19 gene.

63. The population of genetically engineered hematopoietic cells ofembodiment 62, wherein the CD19 gene is an endogenous CD19 gene.

64. The population of genetically engineered hematopoietic cells ofembodiment 62 or 63, wherein the first group of genetically engineeredhematopoietic cells express a mutated CD19 comprising the amino acidsequence of SEQ ID NO: 52 or SEQ ID NO: 73.

65. The population of genetically engineered hematopoietic cells of anyone of embodiments 1-64, wherein at least one of the first and secondlineage-specific cell-surface antigens is a type 2 lineage-specificcell-surface antigen.

66. The population of genetically engineered hematopoietic cells ofembodiment 65, wherein the type 2 lineage-specific cell-surface antigenis CD33.

67. The population of genetically engineered hematopoietic cells ofembodiment 66, wherein the second group of genetically engineeredhematopoietic cells contain genetic editing in exon 2 or exon 3 of aCD33 gene.

68. The population of genetically engineered hematopoietic cells ofembodiment 67, wherein the CD33 gene is an endogenous CD33 gene.

69. The population of genetically engineered hematopoietic cells ofembodiment 67 or 68, wherein the second group of genetically engineeredhematopoietic cells express a mutated CD33 comprising the amino acidsequence of SEQ ID NO: 56 or SEQ ID NO: 58.

70. The population of genetically engineered hematopoietic cells of anyone of embodiments 1-69, which are produced by genomic editing.

71. The population of genetically engineered hematopoietic cells ofembodiment 70, wherein the genome editing involves a zinc fingernuclease (ZFN), a transcription activator-like effector-based nuclease(TALEN), or a CRISPR-Cas system.

72. The population of genetically engineered hematopoietic cells ofembodiment 71, wherein the CRISPR-Cas system comprises a Casendonuclease.

73. The population of genetically engineered hematopoietic cells ofembodiment 72, wherein the Cas endonuclease is a Cas9 endonuclease.

74. The population of genetically engineered hematopoietic cells of anyone of embodiments 1-73, wherein at least one of the first and secondlineage-specific cell-surface antigens is associated with ahematopoietic malignancy.

75. The population of genetically engineered hematopoietic cells of anyone of embodiments 1-74, wherein at least one of the first and secondlineage-specific cell surface antigens is a type 0 protein.

76. The population of genetically engineered hematopoietic cells of anyone of embodiments 1-75, wherein the first and second lineage-specificcell surface antigens are selected from the group consisting of CD7,CD13, CD19, CD20, CD22, CD25, CD32, CD33, CD38, CD44, CD45, CD47, CD56,96, CD117, CD123, CD135, CD174, CLL-1, folate receptor b, IL1RAP, MUC1,NKG2D/NKG2DL, TIM-3, and WT1.

77. The population of genetically engineered hematopoietic cells ofembodiment 76, wherein the first and second lineage-specific cellsurface antigens are selected from the group consisting of:

(i) CD19 and CD33;

(ii) CD19 and CD13;

(iii) CD19 and CD123;

(iv) CD19 and CLL-1;

(v) CD33 and CD13;

(vi) CD33 and CD123;

(vii) CD33 and CLL-1;

(viii) CD13 and CD123;

(ix) CD123 and CLL-1;

(x) CD19, CD33, and CD13;

(xi) CD19, CD33, and CD123;

(xii) CD33, CD13, and CD123;

(xiii) CD19, CD13, and CD123;

(xiv) CLL-1, CD123, and CD33; or

(xv) CD19, CD33, CD13, and CD123.

78. A method for producing genetically engineered hematopoietic cells,the method comprising:

(i) providing a population of hematopoietic cells, and

(ii) genetically editing a first gene encoding a first lineage-specificcell-surface antigen and a second gene encoding a secondlineage-specific cell-surface antigen in the population of hematopoieticto produce the genetically engineered hematopoietic cells.

79. The method of embodiment 78, wherein the first gene and the secondgene are:

(i) CD19 and CD33;

(ii) CD19 and CD13;

(iii) CD19 and CD123;

(iv) CD19 and CLL-1;

(v) CD33 and CD13;

(vi) CD33 and CD123;

(vii) CD33 and CLL-1;

(viii) CD13 and CD123; or

(ix) CD123 and CLL-1.

80. A method for producing genetically engineered hematopoietic cells,the method comprising:

(i) providing a population of hematopoietic cells, and

(ii) genetically editing a CD19 gene, a CD33 gene, or both genes in thepopulation of hematopoietic cells via CRISPR to produce the geneticallyengineered hematopoietic cells,

wherein the genetic editing of the CD19 gene involves one or more guidenucleic acid molecules that target one or more introns of CD19; and

wherein the genetic editing of the CD33 gene involves one or more guidenucleic acid molecules that (a) target one or more introns of CD33;and/or (b) do not target the CD33 pseudogene upstream of the CD33 gene.

81. The method of embodiment 80, wherein the introns of the CD19 genecomprise intron 1 and intron 2, and/or the introns of the CD33 genecomprise intron 1 and intron 2.

82. The method of embodiment 80 or 81, wherein the genetic editing ofCD19 results in exclusion of exon 2 of the CD19 gene; and/or the geneticediting of CD33 results in exclusion of exon 2 of the CD33 gene.

83. A method for producing genetically engineered hematopoietic cells,the method comprising:

(i) providing a population of hematopoietic cells, and

(ii) genetic editing a CD19 gene, a CD33 gene, or both in the populationof hematopoietic cells via CRISPR to produce the genetically engineeredhematopoietic cells,

wherein the genetic editing of the CD19 gene involves at least one guidenucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs:14-26, 67, and 69-72, and/or

wherein the genetic editing of the CD33 gene involves at least one guidenucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs:27-50 and 68.

84. The method of embodiment 83, wherein step (ii) is performed bygenetic editing both a CD19 gene and a CD33 gene in the population ofhematopoietic cells via CRISPR to produce the genetically engineeredhematopoietic cells,

wherein the genetic editing of the CD19 gene involves a guide nucleicacid comprising the nucleotide sequence of SEQ ID NO: 67, and

wherein the genetic editing of the CD33 gene involves a guide nucleicacid comprising the nucleotide sequence of SEQ ID NO: 68.

85. The method of any one of embodiments 80-84, wherein thehematopoietic cells are HSCs.

86. The method of embodiment 85, wherein the HSCs are CD34+/CD33− cells.

87. The method of any one of embodiment 80-86, wherein the hematopoieticcells are from bone marrow cells, cord blood cells, or peripheral bloodmononuclear cells (PBMCs).

88. Genetically engineered hematopoietic cells produced by any one ofembodiments 80-87.

89. A genetically engineered hematopoietic cell, which carries agenetically edited CD19 gene capable of expressing a mutant CD19comprising the amino acid sequence of SEQ ID NO: 52 or 73, and/or agenetically edited CD33 gene capable of expressing a mutant CD33comprising the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 58.

90. The genetically engineered hematopoietic cell of embodiment 89,which carries a genetically edited CD19 gene capable of expressing amutant CD19 comprising the amino acid sequence of SEQ ID NO: 52 or 73,and a genetically edited CD33 gene capable of expressing a mutant CD33comprising the amino acid sequence of SEQ ID NO: 56.

91. A population of genetically engineered hematopoietic stem cells,wherein at least 50% of the hematopoietic stem cells therein carry botha genetically edited CD19 gene and a genetically edited CD33 gene.

92. The population of genetically engineered hematopoietic stem cells ofembodiment 91, wherein the genetically edited CD19 gene is capable ofexpressing a CD19 mutant comprising the amino acid sequence of SEQ IDNO: 52 or 73, and/or the genetically edited CD33 gene is capable ofexpressing a CD33 mutant comprising the amino acid sequence of SEQ IDNO: 56 or SEQ ID NO:58.

93. A genetically engineered hematopoietic cell, wherein exon 2 of CD33gene in the hematopoietic cell is modified and wherein the CD33pseudogene is not modified.

94. The genetically engineered hematopoietic cell of embodiment 93,wherein exon 2 of CD33 gene in the hematopoietic cell is deleted.

95. The genetically engineered hematopoietic cell of embodiment 93 or94, wherein the CD33 pseudogene is not modified by deletion or mutationthat causes a frameshift.

96. The genetically engineered hematopoietic cell of embodiment 93 or94, wherein the CD33 pseudogene is not modified by deletion or mutationthat causes a frameshift in exon 1, intron 1, exon 2, or a combinationthereof.

97. The genetically engineered hematopoietic cell of embodiment 93,wherein the frameshift is in sequence(s) in exon 1, intron 1, and/orexon 2 of the CD33 pseudogene that share sequence homology,respectively, with sequence(s) in exon 1, intron 1, and/or exon 2 ofCD33.

98. A method of supplying hematopoietic cells to a subject (e.g., asubject having a hematopoietic malignancy), comprising:

(a) providing a population of genetically engineered hematopoietic cellsof any one of embodiments 1-77 and 88-97, wherein optionally thegenetically engineered hematopoietic cells comprise HSCs and/or HPCs;and

(b) administering the population of genetically engineered hematopoieticcells to the subject, e.g., under conditions that allow for engraftmentof at least a portion of the population,

thereby supplying the hematopoietic cells to the subject.

99. A method of treating a hematopoietic malignancy, comprisingadministering to a subject in need thereof a population of geneticallyengineered hematopoietic cells of any one of embodiments 1-77 and 88-97.

100. The method of embodiment 99, which further comprises:

-   -   (a) administering to the subject an effective amount of a first        immunotherapeutic agent that targets the first lineage-specific        cell-surface antigen, and    -   (b) administering to the subject an effective amount of a second        immunotherapeutic agent that targets the second lineage-specific        cell-surface antigen.

101. The method of embodiment 100, wherein the first immunotherapeuticagent and the second immunotherapeutic agent are administeredsimultaneously or sequentially (e.g., sequentially with or withoutoverlap, e.g., wherein the first and second immunotherapeutic agent arenot present in the subject at the same time).

102. The method of embodiment 100 or 101, wherein the firstimmunotherapeutic agent is administered when the subject has a primarycancer, and the second immunotherapeutic is administered when thesubject has a relapsed cancer or cancer that is resistant to the firstimmunotherapeutic agent.

103. The method of embodiment 102, wherein the primary cancer is AML andthe relapsed cancer is relapsed AML.

104. The method of embodiment 102, wherein the primary cancer is AML andthe relapsed cancer is ALL.

105. The method of any of embodiments 102 or 104, wherein the relapsedcancer underwent a lineage switch relative to the primary cancer.

106. The method of any of embodiments 102-105, wherein the firstlineage-specific cell surface antigen is absent in the relapsed cancer,or is expressed at a lower level in the relapsed cancer compared to theprimary cancer (e.g., at less than 50%, 40%, 30%, 20%, or 10% of theprotein level in the primary cancer), or is expressed in fewer cancercells in the relapsed cancer compared to the primary cancer (e.g., lessthan 50%, 40%, 30%, 20%, or 10% the relapsed cancer cells detectablyexpress the protein).

107. The method of any of embodiments 102-106, wherein the primarycancer comprises one or more resistant cells, e.g., cells that lack thefirst lineage-specific cell surface antigen or express it at a lowerlevel than in sensitive cells.

108. The method of any of embodiments 89-107, which further comprises:administering to the subject an effective amount of a firstimmunotherapeutic agent that targets expressing the firstlineage-specific cell-surface antigen.

109. The method of embodiment 108, wherein, if the subject experiencesrelapse (e.g., a relapse wherein the cancer is negative for the firstlineage-specific cell surface antigen), then administering to thesubject an effective amount of a second immunotherapeutic agent thattargets the second lineage-specific cell-surface antigen.

110. The method of any of embodiments 100-109, wherein the firstlineage-specific cell-surface antigen is CD19 and the secondlineage-specific cell-surface antigen is CD33.

111. The method of any of embodiments 100-110, wherein the subject has acancer (e.g., a primary cancer) that comprises a first sub-population ofcancer cells and a second sub-population of cancer cells.

112. The method of embodiment 111, wherein the first sub-population ofcancer cells expresses the first lineage-specific cell-surface antigenand the second sub-population of cancer cells expresses the secondlineage-specific cell-surface antigen.

113. The method of embodiment 112, wherein the first sub-population ofcancer cells is targeted by the first immunotherapeutic agent and thesecond sub-population of cancer cells is targeted by the secondimmunotherapeutic agent.

114. The method of embodiment 113, wherein the first sub-population ofcancer cells is resistant to the second immunotherapeutic agent, orwherein the second immunotherapeutic agent is less effective against thefirst sub-population of cancer cells than against the secondsub-population of cancer cells, e.g., by about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90%.

115. The method of embodiment 113 or 114, wherein the secondsub-population of cancer cells is resistant to the firstimmunotherapeutic agent, or wherein the first immunotherapeutic agent isless effective against the second sub-population of cancer cells thanagainst the first sub-population of cancer cells, e.g., by about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

116. The method of any of embodiments 113-115, wherein the firstsub-population of cancer cells does not express the secondlineage-specific cell surface antigen, or the second lineage-specificcell surface antigen is expressed at a lower level in the firstsub-population compared to the second sub-population (e.g., at less than50%, 40%, 30%, 20%, or 10% of the protein level).

117. The method of any of embodiments 113-116, wherein the secondsub-population of cancer cells does not express the firstlineage-specific cell surface antigen, or the first lineage-specificcell surface antigen is expressed at a lower level in the secondsub-population compared to the first sub-population (e.g., at less than50%, 40%, 30%, 20%, or 10% of the protein level).

118. The method of any of embodiments 113-117, wherein the firstsub-population of cancer cells expresses CD33 and the secondsub-population of cancer cells expresses CD123 or CLL-1.

119. The method of any of embodiments 113-118, wherein the firstsub-population of cancer cells is about 50-99%, 60-90%, 70-90%, or about80% of cancer cells in the subject.

120. The method of any of embodiments 113-119, wherein the secondsub-population of cancer cells is about 1-50%, 10-40%, 10-30%, or about20% of cancer cells in the subject.

121. The method of any of embodiments 113-120, wherein the firstsub-population of cancer cells are bulk cancer cells and/or the secondsub-population of cancer cells are cancer stem cells.

122. The method of any of embodiments 113-121, wherein the firstsub-population of cancer cells have one or more markers ofdifferentiated hematopoietic cells and/or the second sub-population ofcancer cells have one or more markers of HSCs or HPCs.

123. The method of any of embodiments 113-122, wherein the firstimmunotherapeutic agent and the second immunotherapeutic agent areadministered simultaneously.

124. The method of any of embodiments 113-123, wherein the firstimmunotherapeutic agent and the second immunotherapeutic agent areadministered such that both of the first immunotherapeutic agent and thesecond immunotherapeutic agent are present in the subject at the sametime.

125. The method of any of embodiments 113-124, further comprisingadministering to the subject an effective amount of a firstimmunotherapeutic agent.

126. The method of embodiment 125, wherein the first immunotherapeuticagent is a cytotoxic agent that targets cells expressing either thefirst lineage-specific cell-surface antigen or the secondlineage-specific cell-surface antigen.

127. The method of embodiment 126, wherein the first immunotherapeuticagent is a cytotoxic agent that targets cells expressing the firstlineage-specific cell-surface antigen, and the method further comprisesadministering to the subject a second immunotherapeutic agent when thehematopoietic malignancy relapses in the subject.

128. The method of embodiment 127, wherein the second immunotherapeuticagent is a cytotoxic agent that targets cells expressing the secondlineage-specific cell-surface antigen.

129. The method of any one of embodiments 126-128, wherein the firstimmunotherapeutic agent, the second immunotherapeutic agent, or both areCAR-T cells.

130. The method of any one of embodiments 126-128, wherein the firstimmunotherapeutic agent, the second immunotherapeutic agent, or both areantibodies.

131. The method of any one of embodiments 126-128, wherein the firstimmunotherapeutic agent, the second immunotherapeutic agent, or both areantibody-drug conjugates.

132. The method of any one of embodiments 113-131, wherein thehematopoietic malignancy is AML.

133. The method of embodiment 132, wherein the AML is relapsed AML.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one or moreof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1B are schematic illustrations showing an example therapeuticprocess involving the methods described herein. FIG. 1A: The processincludes the steps of obtaining CD34+ cells (obtained from a donor orautologously), genetically engineering the CD34+ cells, engrafting theengineered cells into a patient, performing CAR T cell therapy on thepatient, resulting in cleared or reduced cancer burden and retainedhematopoiesis. The sequence corresponds to SEQ ID NO: 57. FIG. 1B: Anengineered donor CD34+ cell in which the non-essential epitope of alineage-specific cell-surface antigen is modified such that it does notbind a CAR T cell that is specific for an epitope of thelineage-specific cell-surface antigen. The sequence corresponds to SEQID NO: 57.

FIG. 2 is a schematic of the extracellular and transmembrane portions ofthe lineage-specific cell-surface protein human CD33. Regions of CD33that are predicted to be less deleterious when modified are indicated bythe boxes. The sequence corresponds to SEQ ID NO: 57.

FIGS. 3A-3B are schematic illustrations showing CAR T cells bind tocells expressing human CD33 but not to cells expressing human CD33 inwhich an epitope of CD33 has been modified or deleted. FIG. 3A: CAR Tcells targeting CD33+ acute myeloid leukemia cells leading to celllysis. The sequence corresponds to SEQ ID NO: 57. FIG. 3B: CAR T cellsare not able to bind to genetically engineered donor graft cells inwhich an epitope of CD33 has been modified or deleted. As a result,these cells do not undergo lysis. The sequence corresponds to SEQ ID NO:57.

FIG. 4 is a schematic of CRISPR/Cas9-mediated genomic deletion of CD19exon 2, resulting in expression of a CD19 variant having exon 2 deleted.

FIGS. 5A-5B include diagrams showing investigation of various modifiedsingle guide RNAs (ms-sgRNAs) targeting CD19 in a human leukemic cellline (K562 cells). FIG. 5A: photos showing PCR amplicons derived fromthe region spanning introns 1 and 2 of the CD19 gene as determined byT7E1 assays. Samples were either treated (+) or untreated (−) with T7E1.The percentage cleavage efficiency is indicated under each lane. C=NewEngland Biolabs™ (NEB™) Sample Control, WT=wild-type untransfectedcells, Cas9=Cas9 only. FIG. 5B: a chart showing the percent INDELdetermined by T7E1 assays and TIDE analysis.

FIGS. 6A-6C include diagrams showing dual ms-sgRNA-mediated deletion ofexon 2 of CD19 in K562 cells. FIG. 6A: a schematic showing a PCR-basedassay to detect CRISPR/Cas9-mediated genomic deletion of exon 2 of CD19via dual ms-sgRNA-mediated CRISPR/Cas9. FIG. 6B: a photo showingdeletion of the region between exon 1 and exon 3 after treating K562cells with indicated pairs of ms-sgRNAs by an end-point PCR assay ofgenomic DNA. FIG. 6C: a chart showing the percentage deletionquantitated by end-point PCR.

FIGS. 7A-7B include diagrams showing screening of CD19 ms-sgRNAstargeting introns 1 or 2 in CD34⁺ HSCs by T7E1 assay and TIDE analysis.FIG. 7A: a photo showing PCR amplicons derived from the region spanningintrons 1 and 2 of the CD19 gene as determined by T7E1 assays. Sampleswere either treated (+) or untreated (−) with T7E1. The percentinsertion/deletion (INDEL) and cleavage efficiency are indicated undereach lane. C=NEB™ Sample Control, Cas9=Cas9 only. FIG. 7B: PCR ampliconsderived from the region spanning introns 1 and 2 of the CD19 gene wereanalyzed by T7E1 Assay or TIDE analysis, and the percent INDEL wasdetermined. Cas9=cas9 only control.

FIGS. 8A-8B include diagrams showing dual ms-sgRNA-mediated deletion ofCD19 exon 2 in CD34+ HSCs. FIG. 8A: a photo showing the smaller deletionPCR product compared to the larger parental band as determined by PCRacross the genomic deletion region. FIG. 8B: a chart showing the percentdeletion quantified by end-point PCR.

FIGS. 9A-9B include diagrams showing investigation of ms-sgRNAstargeting introns 1 or 2 of CD19 in CD34+ HSCs. FIG. 9A: a photo showingPCR amplicons derived from the region spanning introns 1 and 2 of theCD19 gene as determined by T7E1 assays. The percent cleavage efficiencyis indicated under each lane. FIG. 9B: a chart showing PCR ampliconsderived from the region spanning introns 1 and 2 of the CD19 gene asanalyzed by T7E1 assay, and the percent INDEL. Cas9=cas9 only control.

FIGS. 10A-10D include diagrams showing efficient dual ms-sgRNA-mediateddeletion of exon 2 of CD19in CD34+ HSCs. FIG. 10A: a photo showing thesmaller deletion PCR product compared to the larger parental band asdetermined by PCR across the genomic deletion region. The percentdeletion is indicated under each lane. FIG. 10B: a chart showing thepercent deletion quantified by end-point PCR. FIG. 10C: is a photoshowing PCR products of full-length CD19 or CD19 with deletion of exon 2when edited using the pair of gRNA6/gRNA14. FIG. 10D: a chart showingpercentage of CFUs of cells edited by the gRNA6/gRNA14 pair. BFU-E:burst forming unit-erythroid; CFU-GM: colony formingunit-granulocyte/macrophage; CFU-GEMM: colony forming unit ofmultipotential myeloid progenitor cells (generate granulocytes,erythrocytes, monocytes, and megakaryocytes.

FIGS. 11A-11E are diagrams showing production and characterization ofB-cell lymphoma cells having exon 2 deletion in CD19 using a pair ofgRNAs, gRNA6 and gRNA14. FIG. 11A: photos showing PCR products ofgenomic DNA of full-length CD19 and CD19 with exon 2 deletion (leftpanel) and cDNA of full-length CD19 and CD19 with exon 2 deletion. FIG.11B: charts showing quantification of editing efficiency by end-pointPCR. FIG. 11C: a photo showing expressing of full-length CD19 and CD19mutant with deletion of fragment encoded by exon 2 using an antibodyspecific to the C-terminus of CD19. FIG. 11D: charts showing surface andintracellular staining of CD19-expressing in edited cells as measured byflow cytometry. FIG. 11E: charts showing cell number (left panel) andcell viability (right panel) of Raji B-cells expressing CD19exon2.

FIGS. 12A-12C include diagrams illustrating engraftment of CD19ex2hematopoietic stem cells (HSCs) in a mouse model. FIG. 12A: a schematicwork flow to assess differentiation potential of edited CD34+ HSCs.d=days, w=weeks, w/o=week old, RNP=ribonucleoprotein. FIG. 12B: a chartshowing percentage of CD45+ cells in peripheral blood collected frommice engrafted with HSCs expressing CD19ex2. FIG. 12C: a chart showingpercentage of CD19 B cells in peripheral blood collected from miceengrafted with HSCs expressing CD19ex2.

FIG. 13 is a schematic work flow to assess in vivo selectivity andefficacy of CART19 therapy in a Raji Burkitt's lymphoma tumor model.d=days, w=weeks, w/o=week old.

FIGS. 14A-14D include diagrams showing the generation of Raji-fluc-GFPcells in which exon 2 of CD19 has been deleted. FIG. 14A: diagramsshowing expression of CD19 in Raji-fluc-GFP cell lines transfected withthe indicated combinations of ms-sgRNAs as determined by FACS. ParentalRaji cells and Raj-fluc-GFP nucleofected with Cas9 only are included ascontrols. FIG. 14B: is a chart showing the percentage of live cells ineach population of cells (CD19 “hi,” CD19 “int,” and CD19 “lo”). FIG.14C: is a photo showing the smaller PCR product for the exon 2 deletioncompared to the larger parental band as determined by PCR across thegenomic deletion region. FIG. 14D: is a chart showing the percentage ofcells having a deleted exon 2 of CD19 in the bulk population of cells asdetermined by end-point PCR.

FIGS. 15A-15B include diagrams showing the level of CART19 cytotoxicityagainst Raji cells in which CD19 exon 2 has been deleted. FIG. 15A: aline graph showing that cells in which exon 2 of CD19 has been deletedare resistant to CART19 cytotoxicity. FIG. 15B: a bar graph showing thatcells in which exon 2 of CD19 has been deleted are resistant to CART19cytotoxicity.

FIG. 16 is a schematic showing an exemplary in vivo model assessing theefficacy and selectivity of a CART therapeutic paired with edited HSCsinvolving the methods described herein.

FIG. 17 is a schematic showing CD33 exon 2 editing, resulting inexpression of the CD33m variant.

FIGS. 18A-18B include charts showing investigation of various ms-sgRNAstargeting introns 1 or 2 of CD33 in CD34+ HSCs by TIDE analysis. PCRamplicons derived from the region spanning introns 1 and 2 of the CD33gene were analyzed by TIDE analysis and the percent INDEL wasdetermined. FIG. 18A: guide RNAs 1-19. FIG. 18B: guide RNAs 10-24.

FIGS. 19A-19B include diagrams showing characterization of CD33-editedprimary CD34+ HSCs. FIG. 19A: a diagram showing flow cytometric analysisof CD34+ HSCs, either unedited (left panel, mock (“NT”)) or editedproducing a full CD33 knockout (middle panel, “CD33 gRNA KO”), or editedwith the CD33 gRNA-18/gRNA-24 pair resulting in the expression of amutated CD33 with exon 2-encoded fragment deleted (CD33ex2) or producinga full knock out (right panel, “CD33 gRNA 18+24”). FIG. 19B: a chartshowing the percentage of HSCs having CD33 knocked out (“KO”) and CD33with exon 2 deletion (“ex2 Del”) obtained in cells edited by knock-outgRNA or the CD33 gRNA18/gRNA24 pair.

FIGS. 20A-20B include diagrams showing genotyping and in vitrodifferentiation of cells edited by dual gRNAs targeting CD33. FIG. 20A:a chart showing that CD33ex2 cells and CD33KO cells retaineddifferentiation potential in vitro, as determined by a CFU assay. FIG.20B: a photo showing that both CD33ex2 and knock-out alleles wereobserved in differentiated myeloid cells treated with the CD33gRNA18/gRNA24 pair.

FIGS. 21A-21C include diagrams showing generation and characterizationof CD33ex2 in AML cell lines. FIG. 21A: a photo showing genomic PCRresults of selected HL60 clones resulting from gene editing with theCD33 gRNA18/gRNA24 pair. FIG. 21B: a diagram illustrating Taqman™ assaysof total CD33 (Full-length+ex2del, including both full-length and exon 2deletion) and CD33 (with exon 2 deletion). FIG. 21C: charts showing theexpression level of total CD33 (full-length and exon 2 deletion; leftpanel) and the expression level of CD33ex2del in Jurkat cells, parentHL60 cells, and a number of edited HL60 clones.

FIGS. 22A-22C include diagrams showing susceptibility of CD33ex2 cellsto gemtuzumab ozogamicin (GO). FIG. 22A: chart showing viability ofcancer cell lines (Jurkat, THP-1, and HL-60) treated with GO at theindicated concentrations. FIG. 22B: chart showing viability of THP-1cells, CD33ex2 THP-1 cells (generated using CD33 gRNA18/gRNA24 pair),HL-60 cells, and CD33ex2 HL-60 cells (generated using CD33 gRNA18/gRNA24pair) treated with GO at the indicated concentrations. FIG. 22C: chartshowing viability of wild-type HSCs, CD33KO HSCs, and CD33ex2 HSCs(generated using CD33 gRNA18/gRNA24 pair) post-GO treatment (“GO”) ascompared to PBS control (“PBS”).

FIGS. 23A-23D include diagrams showing that CD33ex2 cells are resistantto CART33-mediated cytotoxicity. FIG. 23A: a chart showing the level ofcell lysis of wild-type HL-60 cells (CD33⁺) in the presence of CART33(expressing anti-CD33 CAR1). FIG. 23B: a chart showing the level of celllysis of CD33ex2 HL-60 cells in the presence of CART33. FIG. 23C: achart showing the level of cell lysis of CD33KO HL-60 cells in thepresence of CART33. FIG. 23D: a chart comparing the percentage of celllysis of CD33+ cells, CD33ex2 cells, and CD33KO cells in the presence ofCART33, at the indicated cell ratios.

FIGS. 24A-24D include diagrams showing the results of a TIDE assayshowing efficient multiplex genomic editing of both CD19 and CD33. FIG.24A: a chart showing genomic editing of CD19, CD33, and CD19+CD33 inNALM-6 cells. FIG. 24B: a chart showing genomic editing of CD19, CD33,and CD19+CD33 in HSCs. FIG. 24C: a chart showing genomic editing ofCD19, CD33, and CD19+CD33 in HL-60 cells. FIG. 24D: a chart showinggenomic editing of CD19, CD33, and both CD19 and CD33 in NALM-6 cells.

FIGS. 25A-25C include diagrams showing the results of a nucleofectionassay showing the effect of multiplex genomic editing of both CD19 andCD33 on viability in HSCs and cell lines as compared to single RNAnucleofection. The gRNAs used in the nucleofections are indicated on thex-axis. FIG. 25A: a chart showing percent viability of HSC cellsfollowing genome editing. FIG. 25B: a chart showing percent viability ofNalm-6 cells following genome editing. From left to right, each set ofthree bars corresponds to zero, 24 h, and 48 h. FIG. 25C: a chartshowing percent viability of HL-60 cells following genome editing. Fromleft to right, each set of four bars corresponds to zero, 48 h, 96 h,and 7 d.

FIGS. 26A-26C include diagrams showing sequences and bar graphs of aTIDE analysis of NALM-6 cells transfected with CD19-19 gRNA/RNP andCD33-37 gRNA/RNP complexes. FIG. 26A: a schematic of sequencesidentified and the relative contribution of each sequence for the CD19edited Nalm-6 cells. The sequences from top to bottom correspond to SEQID NOs: 74-86. FIG. 26B: a schematic of sequences identified and therelative contribution of each sequence for the CD33 edited Nalm-6 cells.The sequences from top to bottom correspond to SEQ ID NOs: 87-101. FIG.26C: a chart showing the frequency of INDELS that are +/−1 and +/−2(left columns and right columns, respectively, for each gene). The TIDEanalysis indicates most INDELS are small insertions.

FIG. 27 is a schematic showing the sequence of the CD33 locus includingcut sites for CD33 gRNA-24 and gRNA-18. In this figure, the sequencegtgagtggctgtggggagag (SEQ ID NO: 102) is labeled gRNA-24 and thesequence ttcatgggtactgcagggca (SEQ ID NO: 103) is labeled gRNA-18. Theentire sequence at left can be found in SEQ ID NO: 138, and the entiresequence at right can be found in SEQ ID NO: 139.

FIG. 28 shows an experimental schematic and results showing the editingefficiency achieved in CD34+ HSCs using control (“Mock,” Cas9 only),CD33 knockout (“CD33KO,” CD33 gRNA-37), CD33 exon2 deletion(“CD33ex2del,” CD33 gRNA18 and gRNA-24). The editing efficiency(percentage modification) of the CD33 knockout was assessed by TIDEanalysis, and % INDEL was determined. The fraction population with adeletion of exon 2 was determined by end-point PCR. For the CD33 exon2deletion edited cells, the deletion efficiency of 30% refers to theediting events that resulted in deletion of exon 2, but does not includethe events that resulted in a complete knockout of CD33.

FIG. 29 includes a diagram showing analysis of editing events in HSCsresulting from use of CD33 gRNA-37. TIDE analysis shows the percentageof sequences observed for each INDEL obtained by editing CD34+ HSCsusing gRNA37.

FIG. 30 includes a diagram showing the results of flow cytometricanalysis of unstained cells, unedited (“Mock”), and HSCs edited usingCD33 gRNA-37.

FIGS. 31A-31E include diagrams and a table showing analysis ofpopulations of CD34+ HSCs edited with either CD33 gRNA-37 or the CD33gRNA-18 and gRNA-24 pair, at various times following treatment withgemtuzumab ozogamicin (GO). FIG. 31A: a line graph showing the number ofcells in each of the indicated populations following GO treatment overtime. FIG. 31B: a table showing results corresponding to the graph shownin FIG. 31A. FIG. 31C: a photograph showing analysis of CD33 editingfollowing treatment with gemtuzumab ozogamicin. Percentage of editedcells in the sample edited using CD33 gRNA37 (“KO”) was assessed by TIDEanalysis, and the percentage of edited cells in the sample edited usingCD333 gRNA-18 and gRNA-24 (“CD33ex2del”) was assessed by deletion PCR.FIG. 31D: a chart showing the percent CD14+ cells (myeloiddifferentiation) in the indicated cell populations in the absence ofgemtuzumab ozogamicin over time as indicated. FIG. 31E: a chart showingthe percent CD14+ cells (myeloid differentiation) in the indicated cellpopulations following treatment gemtuzumab ozogamicin over time asindicated.

FIGS. 32A-32B include photographs showing genomic editing of CD19 in theNAM6 cell line transfected with control (“WT,” wildtype/unmodified),CD19 sgRNA 6 and sgRNA-14 pair (“Ex2del-1”), CD19 sgRNA-7 and sgRNA-16pair (“Ex2del-2”), CD19 sgRNA-23 and sgRNA-24 pair (CD19 knock out;“CD19KO”). FIG. 32A: a photograph of a Western blot with an antibodyrecognizing the Ig-like C2-type domain encoded by CD19 exon 4 in theindicated cell populations. FIG. 32B: a photograph of a Western blotwith an antibody recognizing the C-terminus of CD19 in the indicatedcell populations.

FIG. 33 shows target expression on AML cell lines. The expression ofCD33, CD123 and CLL1 in MOLM-13 and THP-1 cells and an unstained controlwas determined by flow cytometric analysis. The X-axis indicates theintensity of antibody staining and the Y-axis corresponds to number ofcells.

FIG. 34 shows CD33− and CD123-modified MOLM-13 cells. The expression ofCD33 and CD123 in wild-type (WT), CD33^(−/−), CD123^(−/−) and CD33^(−/−)CD123^(−/−) MOLM-13 cells was assessed by flow cytometry. For thegeneration of CD33^(−/−) or CD123^(−/−) MOLM-13 cells, WT MOLM-13 cellswere electroporated with CD33− or CD123-targeting RNP, followed by flowcytometric sorting of CD33− or CD123-negative cells.CD33^(−/−)CD123^(−/−) MOLM-13 cells were generated by electroporatingCD33^(−/−) cells with CD123-targeting RNP and sorted for CD123-negativepopulation. The X-axis indicates the intensity of antibody staining andthe Y-axis corresponds to number of cells.

FIG. 35 shows an in vitro cytotoxicity assay of CD33 and CD123 CAR-Ts.Anti-CD33 CAR-T and anti-CD123 CAR-T were incubated with wild-type (WT),CD33^(−/−), CD123^(−/−) and CD33^(−/−) CD123^(−/−) MOLM-13 cells, andcytotoxicity was assessed by flow cytometry. Non-transduced T cells wereused as mock CAR-T control. The CARpool group was composed of 1:1 pooledcombination of anti-CD33 and anti-CD123 CAR-T cells. Student's t testwas used. ns=not significant; *P<0.05; **P<0.01. The Y-axis indicatesthe percentage of specific killing.

FIG. 36 shows CD33− and CLL1-modified HL-60 cells. The expression ofCD33 and CLL1 in wild-type (WT), CD33^(−/−), CLL1^(−/−) and CD33^(−/−)CLL1^(−/−) HL-60 cells was assessed by flow cytometry. For thegeneration of CD33^(−/−) or CLL1^(−/−) HL-60 cells, WT HL-60 cells wereelectroporated with CD33− or CLL1-targeting RNP, followed by flowcytometric sorting of CD33− or CLL1-negative cells. CD33^(−/−)CLL1^(−/−)HL-60 cells were generated by electroporating CD33^(−/−) cells withCLL1-targeting RNP and sorted for CLL1-negative population. The X-axisindicates the intensity of antibody staining and the Y-axis correspondsto number of cells.

FIG. 37 shows an in vitro cytotoxicity assay of CD33 and CLL1 CAR-Ts.Anti-CD33 CAR-T and anti-CLL1 CAR-T were incubated with wild-type (WT),CD33^(−/−), CLL1^(−/−) and CD33^(−/−) CLL1^(−/−) HL-60 cells, andcytotoxicity was assessed by flow cytometry. Non-transduced T cells wereused as mock CAR-T control. The CARpool group was composed of 1:1 pooledcombination of anti-CD33 and anti-CLL1 CAR-T cells. Student's t test wasused. ns=not significant; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001.The Y-axis indicates the percentage of specific killing.

FIG. 38 shows gene-editing efficiency of CD34+ cells. Human CD34+ cellswere electroporated with Cas9 protein and CD33−, CD123− or CLL1−targeting gRNAs, either alone or in combination. Editing efficiency ofCD33, CD123 or CLL1 locus was determined by Sanger sequencing and TIDEanalysis. The Y-axis indicates the editing efficiency (% by TIDE).

FIGS. 39A-39C show in vitro colony formation of gene-edited CD34+ cells.Control or CD33, CD123, CLL-1-modified CD34+ cells were plated inMethoCult™ 2 days after electroporation and scored for colony formationafter 14 days. BFU-E: burst forming unit-erythroid; CFU-GM: colonyforming unit-granulocyte/macrophage; CFU-GEMM: colony forming unit ofmultipotential myeloid progenitor cells (generate granulocytes,erythrocytes, monocytes, and megakaryocytes). Student's t test was used.

DETAILED DESCRIPTION OF THE INVENTION

Successfully identifying suitable proteins for targeted cancer therapiespresents a significant challenge. Many potential target proteins arepresent on both the cell surface of a cancer cell and on the cellsurface of normal, non-cancer cells, which may be required or criticallyinvolved in the development and/or survival of the subject. Many of thetarget proteins contribute to the functionality of such essential cells.Thus, therapies targeting these proteins may lead to deleterious effectsin the subject, such as significant toxicity and/or other side effects.Further, resistance to CAR-T therapy remains a challenge in treatment ofhematopoietic malignance, such as acute myeloid leukemia (AML) due toswitch of cancer antigens on cancer cells, thereby escaping CAR-Ttherapy. For example, patients having B-cell acute lymphoblasticleukemia (B-ALL) were found to develop acute myeloid leukemia (AML) withCD19⁻ cancer cells after CAR-T therapy.

The present disclosure provides methods, cells, compositions, and kitsaimed at addressing at least the above-stated problems. The methods,cells, compositions, and kits described herein provide a safe andeffective treatment for hematological malignancies, allowing fortargeting of one or more lineage-specific cell surface proteins (e.g.,type 0, type 1, or type 2 proteins) that are present not only on cancercells but also on cells critical for the development and/or survival ofthe subject.

More specifically, hematopoietic cells as described herein can be used(for example) in the treatment of a subject that receives two or moredifferent therapies for cancer. Many therapies deplete the subject'sendogenous, non-cancerous hematopoietic cells. Replacement or rescuehematopoietic cells described herein can replace the subject's depletedimmune cells. Two particular examples of this method are describedbelow.

First, in some cases, a subject receives the first therapy (e.g.,against CD19), and then the cancer relapses, and then the subjectreceives the second therapy (e.g., against CD33). The presentapplication provides, e.g., rescue cells that are resistant to boththerapies. Thus, the rescue cells can be administered to the subject ator near the time of the first therapy, and if relapse occurs, thesubject can then receive the second therapy without depleting the rescuecells.

Second, in some cases, a subject may need to receive two therapies atonce, e.g., because the cancer comprises two sub-populations of cells(e.g., one expressing CD33 and the second expressing CD123 and/orCLL-1), and each therapy only attacks one of the sub-populations. Asdescribed herein, rescue cells resistant to both therapies can replacethe subject's depleted immune cells even in the presence of boththerapies.

Experimental evidence provided in the working Examples hereindemonstrates the production, viability, differentiation potential, andresistance to therapy of various cells edited at two antigens. Forinstance, Examples 3 and 4 show a high frequency of multiplex editing ofCD19 and CD33 HSC cells, without impairing viability. Example 9 shows ahigh frequency of editing of other pairs of cell surface antigens, e.g.,CD33 and CD123, CD33 and CLL1, and CD123 and CLL1. Example 9 also showsthat doubly edited cells show resistance to CART targeting the antigens.

The present disclosure also provides a population of rescuehematopoietic cells that comprises a first sub-population of cells thatis (and/or gives rise to) cells resistant to a first therapy and asecond sub-population of cells that is (and/or gives rise to) cellsresistant to a second therapy. (Optionally, the population can comprisecells that are (and/or give rise to) cells resistant to both therapies;however this is not required in this embodiment). The cell populationscan be useful, e.g., when subjects are treated with two therapiessequentially. For instance, in some embodiments, the edited cell-surfaceantigens are antigens that are typically not expressed in normal HSCs,but become expressed in later lineages, so the transplanted HSCs areresistant to both therapies regardless of whether any HSCs are editedfor both antigens. This population of HSCs will continue to producedifferentiated cells, some of which are deficient for the first antigen,and some of which are deficient for the second antigen. When the subjectis treated with the first therapy, differentiated cells deficient forthe first antigen will survive, and when the subject is treated with thesecond therapy, differentiated cells deficient for the second antigenwill survive. Thus, such heterogeneous populations of cells can beuseful as rescue cells.

Accordingly, described herein are genetically engineered hematopoieticcells such as hematopoietic stem cells (HSCs) having genetic editing inone or more genes coding for lineage-specific cell-surface proteins, forexample, CD33 and/or CD19; methods of producing such, for examples, viathe CRISPR approach using specific guide RNAs; and methods of treating ahematopoietic malignancy using the engineered hematopoietic cells,either taken alone, or in combination with one or more cytotoxic agents(e.g., CAR-T cells) that can target the wild-type lineage-specificcell-surface antigens but not those encoded by the edited genes in theengineered hematopoietic cells.

I. Genetically Engineered Hematopoietic Cells

The present disclosure provides genetically engineered hematopoieticcells such as hematopoietic stem cells that carry genetically editedgenes for reducing or eliminating expression of one or morelineage-specific cell-surface antigens, or for expressing the one ormore lineage-specific cell-surface antigens in mutated form. The mutatedantigens would retain at least partial bioactivity of the antigens butcan escape targeting by cytotoxic agents such as CAR-T cells specific tothe antigens. In some embodiments, the lineage-specific cell-surfaceantigens of interest may not be expressed on hematopoietic cells such asHSCs in nature. However, such antigens may be expressed on cellsdifferentiated from the HSCs (e.g., descendants thereof). “Expressing alineage-specific cell-surface protein” or “expressing a lineage-specificcell-surface antigen” means that at least a portion of thelineage-specific cell-surface protein, or antigen thereof, can bedetected on the surface of the hematopoietic cells or descendantsthereof. As used herein, “descendants” of hematopoietic cells includeany cell type or lineage of cells that arise from the hematopoieticcells. In some embodiments, the descendants of the hematopoietic cellsare a cell type or lineage of cells that have differentiated from thehematopoietic cells.

The genetically engineered hematopoietic cells may be used alone fortreating hematopoietic malignancies, or in combination with one or morecytotoxic agents that target the wild-type lineage-specific cell-surfaceantigens but not the mutant encoded by the edited genes in thegenetically engineered hematopoietic cells. Such hematopoietic cells,upon differentiation, could compensate the loss of function caused byelimination of functional non-cancerous cells due to immunotherapy thattargets lineage-specific cell-surface antigen(s), which may alsoexpressed on normal cells. This approach would also broaden the choiceof target proteins for immunotherapy such as CART therapy. For example,certain lineage-specific cell-surface proteins (e.g., Type 0 antigen)are essential to the development and/or survival of essentialcells/tissue and thus are poor target in conventional immunotherapy.Being compensated by the genetically engineered hematopoietic stem cellsdescribed herein, such lineage-specific cell-surface proteins (e.g.,Type 0 antigen) could also be suitable targets of immunotherapy, when itis combined with the engineered HSCs.

(A) Genetically Engineered Hematopoietic Cells Expressing MultipleLineage-Specific Cell Surface Antigens in Mutated Form

In some embodiments, provided herein are a population of geneticallyengineered hematopoietic cells such as HSCs, which collectively carrygenetically edited genes of at least two lineage-specific cell-surfaceproteins. The genetically edited genes express the antigens in mutatedform, e.g., having one or more non-essential epitopes deleted or mutatedso as to escape recognition (e.g., have a reduced binding activity) bycytotoxic agents specific to the antigens. Deletion or mutation of anon-essential epitope in a lineage-specific cell-surface protein is notexpected to dramatically affect the biological activity of such anantigen.

In some instances, the hematopoietic cell population (e.g., HSCs)described herein can be homogenous, including cells each carryingmultiple genetically edited genes (e.g., 2, 3, or 4) of lineage-specificcell-surface antigens. In other instances, the hematopoietic cellpopulation is heterogeneous, comprising (a) cells that carry agenetically edited gene encoding a first lineage-specific cell-surfaceantigen, (b) cells that carry a genetically edited gene encoding asecond lineage-specific cell-surface antigen (which is different fromthe first antigen), and/or (c) cells that carrying genetically editedgenes of both the first and second lineage-specific cell-surfaceantigens.

In some embodiments, the population of cells obtained post editingcomprises cells that have one or more of the target genes partially orcompletely deleted or both. In some embodiments, the population of cellsobtained post editing comprise cells which have gene(s) encoding one ormore lineage-specific antigen(s) which are edited such that expressionresults in a lineage-specific antigen(s) having a partial sequencedeletion, e.g., lacking one or more exon(s) of the lineage-specificantigen, and cells comprising edited gene(s) which result in a completeKO of the lineage-specific antigen. In some embodiments, the populationof cells obtained post editing comprise cells which have gene(s)encoding one or more lineage-specific antigen(s) which are edited suchthat expression results in a lineage-specific antigen(s) having apartial sequence deletion, e.g., lacking one or more exon(s) of thelineage-specific antigen, and also have edited gene(s) which result in acomplete KO of the lineage-specific antigen.

In some embodiments, the population of cells obtained post editingcomprise cells which have gene(s) encoding CD19 and/or CD33 which areedited such that expression results in a CD19 and/or CD33 having apartial sequence deletion, e.g., lacking one or more exon(s) of CD19and/or CD33, and cells comprising edited CD19 and/or CD33 gene(s) whichresult in a complete KO of the lineage-specific antigen. In someembodiments, the population of cells obtained post editing comprisecells which have gene(s) encoding CD19 and/or CD33 which are edited suchthat expression results in a CD19 and/or CD33 polypeptide having apartial sequence deletion, e.g., lacking one or more exon(s) of CD19and/or CD33, and also have edited gene(s) which result in a complete KOof CD19 and/or CD33.

Lineage-Specific Cell-Surface Proteins

As used herein, the terms “protein,” “peptide,” and “polypeptide” may beused interchangeably and refer to a polymer of amino acid residueslinked together by peptide bonds. In general, a protein may be naturallyoccurring, recombinant, synthetic, or any combination of these. Alsowithin the scope of the term are variant proteins, which comprise amutation (e.g., substitution, insertion, or deletion) of one or moreamino acid residues relative to the wild-type counterpart.

As used herein, the terms “lineage-specific cell-surface protein” and“cell-surface lineage-specific protein” may be used interchangeably andrefer to any protein that is sufficiently present on the surface of acell and is associated with one or more populations of cell lineage(s).For example, the protein may be present on one or more populations ofcell lineage(s) and absent (or at reduced levels) on the cell-surface ofother cell populations. In some embodiments, the terms lineage-specificcell-surface antigen” and “cell-surface lineage-specific antigen” maybeused interchangeably and refer to any antigen of a lineage-specificcell-surface protein.

In general, lineage-specific cell-surface proteins can be classifiedbased on a number of factors, such as whether the protein and/or thepopulations of cells that present the protein are required for survivaland/or development of the host organism. A summary of exemplary types oflineage-specific proteins is provide in Table 1 below.

TABLE 1 Classification of Lineage Specific Proteins Type of LineageSpecific Protein Characteristics of the Lineage Specific Protein Type 0a) protein is required for survival of an organism, and b) cell typecarrying type 0 protein is required for survival of an organism and isnot unique to a tumor, or tumor-associated virus Type 1 a) protein isnot required for survival of an organism, and b) cell type carrying type1 protein is not required for survival of an organism Type 2 a) proteinis not required for survival of an organism, and b) cell type carryingtype 2 protein is required for the survival of an organism Type 3 a)protein is not required for the survival of an organism; b) cell typecarrying protein is not required for survival of an organism; and c) Theprotein is unique to a tumor, or a tumor associated virus An example isthe LMP-2 protein in EBV infected cells, including EBV infected tumorcells (Nasopharyngeal carcinoma and Burkitts Lymphoma)

As shown in Table 1, type 0 lineage-specific cell-surface proteins arenecessary for the tissue homeostasis and survival, and cell typescarrying type 0 lineage-specific cell-surface protein may be alsonecessary for survival of the subject. Thus, given the importance oftype 0 lineage-specific cell-surface proteins, or cells carrying type 0lineage-specific cell-surface proteins, in homeostasis and survival,targeting this category of proteins may be challenging usingconventional CAR T cell immunotherapies, as the inhibition or removal ofsuch proteins and cell carrying such proteins may be detrimental to thesurvival of the subject. Consequently, lineage-specific cell-surfaceproteins (such as type 0 lineage-specific proteins) and/or the celltypes that carry such proteins may be required for the survival, forexample because it performs a vital non-redundant function in thesubject, then this type of lineage specific protein may be a poor targetfor conventional CAR T cell based immunotherapies.

However, by combining the genetically engineered hematopoietic stemcells described herein and cytotoxic agent such as CAR-T cell-basedtherapy, the selection of target antigen can be expanded to essentialantigens such as type 0 lineage-specific cell-surface proteins. In someembodiments, the engineered hematopoietic cells (e.g., HSCs) have one ormore genes of type 0 antigens edited for expression of these type 0antigens in mutated form, which retain (at least partially) bioactivityof the type 0 antigens but can escape targeting by type 0antigen-specific cytotoxic agents such as CAR-T cells so as to remedythe loss of normal cells expressing the type 0 antigens due to thetherapy.

In contrast to type 0 proteins, type 1 cell-surface lineage-specificproteins and cells carrying type 1 cell-surface lineage-specificproteins are not required for tissue homeostasis or survival of thesubject. Targeting type 1 cell-surface lineage-specific proteins is notlikely to lead to acute toxicity and/or death of the subject. Forexample, as described in Elkins et al. (Mol. Cancer Ther. (2012)10:2222-32) a CAR T cell engineered to target CD307, a type 1 proteinexpressed uniquely on both normal plasma cells and multiple myeloma (MM)cells would lead to elimination of both cell types. However, since theplasma cell lineage is expendable for the survival of the organism,CD307 and other type 1 lineage specific proteins are proteins that aresuitable for CAR T cell based immunotherapy. Lineage specific proteinsof type 1 class may be expressed in a wide variety of different tissues,including, ovaries, testes, prostate, breast, endometrium, and pancreas.

In some embodiments, the genetically engineered hematopoietic cells(e.g., HSCs) have one or more genes of type 1 antigens for expression ofthe type 1 proteins in mutated forms, which retain (at least partially)bioactivity of the type 1 antigens but can escape targeting by type 1antigen-specific cytotoxic agents such as CAR-T cells. Use of suchengineered HSCs (either alone or in combination with cytotoxic agentssuch as CAR-T cells targeting the type 1 antigens) may improve thelonger-term survival and quality of life of the patient. For example,targeting all plasma cells, while not expected to lead to acute toxicityand/or death, could have longer-term consequences such as reducedfunction of the humoral immune system leading to increased risk ofinfection.

Targeting type 2 proteins presents a significant difficulty as comparedto type 1 proteins. Type 2 proteins are those characterized where: (1)the protein is dispensable for the survival of an organism (i.e., is notrequired for the survival), and (2) the cell lineage carrying theprotein is indispensable for the survival of an organism (i.e., theparticular cell lineage is required for the survival). For example, CD33is a type 2 protein expressed in both normal myeloid cells as well as inAcute Myeloid Leukemia (AML) cells (Dohner et al., NEJM 373:1136(2015)). As a result, a CART cell engineered to target CD33 proteincould lead to the killing of both normal myeloid cells as well as AMLcells, which may be incompatible with survival of the subject.

In some embodiments, the genetically engineered hematopoietic cells(e.g., HSCs) have one or more genes of type 2 antigens for expression ofthe type 2 antigens in mutated form, which retain (at least partially)bioactivity of the type 2 antigens but can escape targeting by type 1antigen-specific cytotoxic agents such as CAR-T cells. Use of suchengineered HSCs (either alone or in combination with cytotoxic agentssuch as CAR-T cells targeting the type 2 antigens) may improve thelonger-term survival and quality of life of the patient. For example,targeting all plasma cells, while not expected to lead to acute toxicityand/or death, could have longer-term consequences such as reducedfunction of the humoral immune system leading to increased risk ofinfection.

In some embodiments, the cell-surface lineage-specific protein is BCMA,CD19, CD20, CD30, ROR1, B7H6, B7H3, CD23, CD38, C-type lectin likemolecule-1, CS1, IL-5, L1-CAM, PSCA, PSMA, CD138, CD133, CD70, CD7,CD13, NKG2D, NKG2D ligand, CLEC12A, CD11, CD123, CD56, CD34, CD14, CD33,CD66b, CD41, CD61, CD62, CD235a, CD146, CD326, LMP2, CD22, CD52, CD10,CD3/TCR, CD79/BCR, and CD26. In some embodiments, the cell-surfacelineage-specific protein is CD33 or CD19.

Alternatively or in addition, the cell-surface lineage-specific proteinmay be a cancer protein, for example a cell-surface lineage-specificprotein that is differentially present on cancer cells. In someembodiments, the cancer protein is a protein that is specific to atissue or cell lineage. Examples of cell-surface lineage-specificprotein that are associated with a specific type of cancer include,without limitation, CD20, CD22 (Non-Hodgkin's lymphoma, B-cell lymphoma,chronic lymphocytic leukemia (CLL)), CD52 (B-cell CLL), CD33 (Acutemyelogenous leukemia (AML)), CD10 (gp100) (Common (pre-B) acutelymphocytic leukemia and malignant melanoma), CD3/T-cell receptor (TCR)(T-cell lymphoma and leukemia), CD79/B-cell receptor (BCR) (B-celllymphoma and leukemia), CD26 (epithelial and lymphoid malignancies),human leukocyte antigen (HLA)-DR, HLA-DP, and HLA-DQ (lymphoidmalignancies), RCAS1 (gynecological carcinomas, biliary adenocarcinomasand ductal adenocarcinomas of the pancreas) as well as prostate specificmembrane antigen. In some embodiments, the cell-surface protein CD33 andis associated with AML cells.

In some embodiments, the genetically engineered HSCs may have editedgenes which encode at least two (e.g., two, three, or four)lineage-specific cell-surface proteins, which can be selected from CD7,CD13, CD19, CD22, CD20, CD25, CD32, CD33, CD38, CD44, CD45, CD47, CD56,96, CD117, CD123, CD135, CD174, CLL-1, folate receptor β, IL1RAP, MUC1,NKG2D/NKG2DL, TIM-3, and WT1. In specific examples, the geneticallyengineered HSCs may have edited genes of the following combinations: (a)CD19+CD33, (b) CD19+CD13, (c) CD19+CD123, (d) CD33+CD13, (e) CD33+CD123,(f) CD13+CD123, (g) CD19+CD33+CD13, (h) CD19+CD33+CD123, (i)CD19+CD13+CD123, (j) CD33+CD13+CD123, or (k) CD19+CD33+CD13+CD123. Insome embodiments, the genetically engineered HSCs may have edited geneswhich encode CD33 and one or more lineage-specific cell-surface proteinsselected from CD7, CD13, CD19, CD22, CD20, CD25, CD32, CD33, CD38, CD44,CD45, CD47, CD56, 96, CD117, CD123, CD135, CD174, CLL-1, folate receptor(3, IL1RAP, MUC1, NKG2D/NKG2DL, TIM-3, and WT1.

In some embodiments, one or both of the lineage-specific cell surfaceproteins are chosen from CD1a, CD1b, CD1c, CD1 d, CD1e, CD2, CD3, CD3 d,CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b,CD11c, CD11d, CDw12, CD13, CD14, CD15, CD16, CD16b, CD17, CD18, CD19,CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31,CD32a, CD32b, CD32c, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40,CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB,CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e,CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59,CD60a, CD61, CD62E, CD62L, CD62P, CD63, CD64a, CD65, CD65s, CD66a,CD66b, CD66c, CD66F, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75,CD75S, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85A, CD85C,CD85D, CD85E, CD85F, CD85G, CD85H, CD85I, CD85J, CD85K, CD86, CD87,CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99,CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b,CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117,CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD121a, CD121b, CD122,CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD131, CD132, CD133,CD134, CD135, CD136, CD137, CD138, CD139, CD140a, CD140b, CD141, CD142,CD143, CD14, CDw145, CD146, CD147, CD148, CD150, CD152, CD152, CD153,CD154, CD155, CD156a, CD156b, CD156c, CD157, CD158b1, CD158b2, CD158 d,CD158e1/e2, CD158f, CD158g, CD158 h, CD158i, CD158j, CD158k, CD159a,CD159c, CD160, CD161, CD163, CD164, CD165, CD166, CD167a, CD168, CD169,CD170, CD171, CD172a, CD172b, CD172g, CD173, CD174, CD175, CD175s,CD176, CD177, CD178, CD179a, CD179b, CD180, CD181, CD182, CD183, CD184,CD185, CD186, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CDw198,CDw199, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD207, CD208,CD209, CD210a, CDw210b, CD212, CD213a1, CD213a2, CD215, CD217, CD218a,CD218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228,CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD236, CD236R,CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247,CD248, CD249, CD252, CD253, CD254, CD256, CD257, CD258, CD261, CD262,CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD272, CD272,CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282,CD283, CD284, CD286, CD288, CD289, CD290, CD292, CDw293, CD294, CD295,CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303,CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307 d, CD307e, CD309,CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322,CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334,CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351,CD352, CD353, CD354, CD355, CD357, CD358, CD359, CD360, CD361, CD362 andCD363.

In some embodiments, one or both of the lineage-specific cell surfaceproteins are chosen from CD19; CD123; CD22; CD30; CD171; CS-1 (alsoreferred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-typelectin-like molecule-1 (CLECL1); CD33; epidermal growth factor receptorvariant III (EGFRvIII); ganglioside G2 (CD2); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlep(1-1)Cer); TNF receptor familymember B cell maturation (BCMA), Tn antigen ((Tn Ag) or(GalNAc.alpha.-Ser/Thr)); prostate-specific membrane antigen (PSMA);Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-Liketyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72);CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesionmolecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptorsubunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); ProteaseSerine 21 (Testisin or PRSS21); vascular endothelial growth factorreceptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growthfactor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4(SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinaseERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1); epidermalgrowth factor receptor (EGFR); neural cell adhesion molecule (NCAM);Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated(ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP);insulin-like growth factor I receptor (IGF-I receptor), carbonicanhydrase IX (CAIX), Proteasome (Prosome, Macropain) Subunit, Beta Type9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consistingof breakpoint cluster region (BCR) and Abelson murine leukemia viraloncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2(EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); gangliosideGM3 (aNeu5Ac(2-3)bDGalp(1-4)bDG1cp(1-1)Cer); transglutaminase 5 (TGS5);high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1(TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6(CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupledreceptor class C group 5, member D (GPRC5D); chromosome X open readingframe 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK);Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion ofgloboH glycoceramide (GloboH); mammary gland differentiation antigen(NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1(HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); Gprotein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex; locusK 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma AlternateReading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testisantigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-1a);Melanoma-associated antigen 1 (MAGE-A1), ETS translocation-variant gene6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); XAntigen Family, member 1A (XAGE1); angiopoietin-binding cell surfacereceptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1;tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase;prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanomaantigen recognized by T cells 1 (MelanA or MART1); Rat sarcoma (Ras)mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-1AP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viraloncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family MemberC (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1(CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS orBrother of the Regulator of Imprinted Sites), Squamous Cell CarcinomaAntigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5(PAXS); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced GlycationEndproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2(RU2); legumain; human papilloma virus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxy esterase; heat shock protein 70-2mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associatedimmunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor(FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily Amember 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-typelectin domain family 12 member A (CLEC12A); bone marrow stromal cellantigen 2 (BST2); EGF-like module-containing mucin-like hormonereceptor-like 2 (EMR2), lymphocyte antigen 75 (LY75); Glypican-3 (GPC3);Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1(IGLL1).

In some embodiments, one or both of the lineage-specific cell surfaceproteins are chosen from CD11a, CD18, CD19, CD20, CD31, CD34, CD44,CD45, CD47, CD51, CD58, CD59, CD63, CD97, CD99, CD100, CD102, CD123,CD127, CD133, CD135, CD157, CD172b, CD217, CD300a, CD305, CD317, CD321,CD33, and CLL1.

In some embodiments, one or both of the lineage-specific cell surfaceproteins are chosen from CD33, CD123, CLL1, CD38, CD135 (FLT3), CD56(NCAM1), CD117 (c-KIT), FRβ (FOLR2), CD47, CD82, TNFRSF1B (CD120B),CD191, CD96, PTPRJ (CD148), CD70, LILRB2 (CD85D), CD25 (IL2Ralpha),CD44, CD96, NKG2D Ligand, CD45, CD7, CD15, CD19, CD20, CD22, CD37, andCD82.

In some embodiments, one or both of the lineage-specific cell surfaceproteins are chosen from CD7, CD11a, CD15, CD18, CD19, CD20, CD22, CD25,CD31, CD33, CD34, CD37, CD38, CD44, CD45, CD47, CD51, CD56, CD58, CD59,CD63, CD70, CD82, CD85D, CD96, CD97, CD99, CD100, CD102, CD117, CD120B,CD123, CD127, CD133, CD135, CD148, CD157, CD172b, CD191, CD217, CD300a,CD305, CD317, CD321, CLL1, FRβ (FOLR2), NKG2D Ligand.

Table 1A lists exemplary pairs of first and second lineage-specific cellsurface proteins that can be used in accordance with the compositionsand methods described herein. — CD11a, CD7 CD15, CD7 CD18, CD7 CD19, CD7CD7, CD11a — CD15, CD11a CD18, CD11a CD19, CD11a CD7, CD15 CD11a, CD15 —CD18, CD15 CD19, CD15 CD7, CD18 CD11a, CD18 CD15, CD18 — CD19, CD18 CD7,CD19 CD11a, CD19 CD15, CD19 CD18, CD19 — CD7, CD20 CD11a, CD20 CD15,CD20 CD18, CD20 CD19, CD20 CD7, CD22 CD11a, CD22 CD15, CD22 CD18, CD22CD19, CD22 CD7, CD25 CD11a, CD25 CD15, CD25 CD18, CD25 CD19, CD25 CD7,CD31 CD11a, CD31 CD15, CD31 CD18, CD31 CD19, CD31 CD7, CD33 CD11a, CD33CD15, CD33 CD18, CD33 CD19, CD33 CD7, CD34 CD11a, CD34 CD15, CD34 CD18,CD34 CD19, CD34 CD7, CD37 CD11a, CD37 CD15, CD37 CD18, CD37 CD19, CD37CD7, CD38 CD11a, CD38 CD15, CD38 CD18, CD38 CD19, CD38 CD7, CD44 CD11a,CD44 CD15, CD44 CD18, CD44 CD19, CD44 CD7, CD45 CD11a, CD45 CD15, CD45CD18, CD45 CD19, CD45 CD7, CD47 CD11a, CD47 CD15, CD47 CD18, CD47 CD19,CD47 CD7, CD51 CD11a, CD51 CD15, CD51 CD18, CD51 CD19, CD51 CD7, CD56CD11a, CD56 CD15, CD56 CD18, CD56 CD19, CD56 CD7, CD58 CD11a, CD58 CD15,CD58 CD18, CD58 CD19, CD58 CD7, CD59 CD11a, CD59 CD15, CD59 CD18, CD59CD19, CD59 CD7, CD63 CD11a, CD63 CD15, CD63 CD18, CD63 CD19, CD63 CD7,CD70 CD11a, CD70 CD15, CD70 CD18, CD70 CD19, CD70 CD7, CD82 CD11a, CD82CD15, CD82 CD18, CD82 CD19, CD82 CD7, CD85D CD11a, CD85D CD15, CD85DCD18, CD85D CD19, CD85D CD7, CD96 CD11a, CD96 CD15, CD96 CD18, CD96CD19, CD96 CD7, CD97 CD11a, CD97 CD15, CD97 CD18, CD97 CD19, CD97 CD7,CD99 CD11a, CD99 CD15, CD99 CD18, CD99 CD19, CD99 CD7, CD100 CD11a,CD100 CD15, CD100 CD18, CD100 CD19, CD100 CD7, CD102 CD11a, CD102 CD15,CD102 CD18, CD102 CD19, CD102 CD7, CD117 CD11a, CD117 CD15, CD117 CD18,CD117 CD19, CD117 CD7, CD120B CD11a, CD120B CD15, CD120B CD18, CD120BCD19, CD120B CD7, CD123 CD11a, CD123 CD15, CD123 CD18, CD123 CD19, CD123CD7, CD127 CD11a, CD127 CD15, CD127 CD18, CD127 CD19, CD127 CD7, CD133CD11a, CD133 CD15, CD133 CD18, CD133 CD19, CD133 CD7, CD135 CD11a, CD135CD15, CD135 CD18, CD135 CD19, CD135 CD7, CD148 CD11a, CD148 CD15, CD148CD18, CD148 CD19, CD148 CD7, CD157 CD11a, CD157 CD15, CD157 CD18, CD157CD19, CD157 CD7, CD172b CD11a, CD172b CD15, CD172b CD18, CD172b CD19,CD172b CD7, CD191 CD11a, CD191 CD15, CD191 CD18, CD191 CD19, CD191 CD7,CD217 CD11a, CD217 CD15, CD217 CD18, CD217 CD19, CD217 CD7, CD300aCD11a, CD300a CD15, CD300a CD18, CD300a CD19, CD300a CD7, CD305 CD11a,CD305 CD15, CD305 CD18, CD305 CD19, CD305 CD7, CD317 CD11a, CD317 CD15,CD317 CD18, CD317 CD19, CD317 CD7, CD321 CD11a, CD321 CD15, CD321 CD18,CD321 CD19, CD321 CD7, CLL1 CD11a, CLL1 CD15, CLL1 CD18, CLL1 CD19, CLL1CD7, FOLR2 CD11a, FOLR2 CD15, FOLR2 CD18, FOLR2 CD19, FOLR2 CD7, NKG2DCD11a, NKG2D CD15, NKG2D CD18, NKG2D CD19, NKG2D Ligand Ligand LigandLigand Ligand CD7, EMR2 CD11a, EMR2 CD15, EMR2 CD18, EMR2 CD19, EMR2CD20, CD7 CD22, CD7 CD25, CD7 CD31, CD7 CD33, CD7 CD20, CD11a CD22,CD11a CD25, CD11a CD31, CD11a CD33, CD11a CD20, CD15 CD22, CD15 CD25,CD15 CD31, CD15 CD33, CD15 CD20, CD18 CD22, CD18 CD25, CD18 CD31, CD18CD33, CD18 CD20, CD19 CD22, CD19 CD25, CD19 CD31, CD19 CD33, CD19 —CD22, CD20 CD25, CD20 CD31, CD20 CD33, CD20 CD20, CD22 — CD25, CD22CD31, CD22 CD33, CD22 CD20, CD25 CD22, CD25 — CD31, CD25 CD33, CD25CD20, CD31 CD22, CD31 CD25, CD31 — CD33, CD31 CD20, CD33 CD22, CD33CD25, CD33 CD31, CD33 — CD20, CD34 CD22, CD34 CD25, CD34 CD31, CD34CD33, CD34 CD20, CD37 CD22, CD37 CD25, CD37 CD31, CD37 CD33, CD37 CD20,CD38 CD22, CD38 CD25, CD38 CD31, CD38 CD33, CD38 CD20, CD44 CD22, CD44CD25, CD44 CD31, CD44 CD33, CD44 CD20, CD45 CD22, CD45 CD25, CD45 CD31,CD45 CD33, CD45 CD20, CD47 CD22, CD47 CD25, CD47 CD31, CD47 CD33, CD47CD20, CD51 CD22, CD51 CD25, CD51 CD31, CD51 CD33, CD51 CD20, CD56 CD22,CD56 CD25, CD56 CD31, CD56 CD33, CD56 CD20, CD58 CD22, CD58 CD25, CD58CD31, CD58 CD33, CD58 CD20, CD59 CD22, CD59 CD25, CD59 CD31, CD59 CD33,CD59 CD20, CD63 CD22, CD63 CD25, CD63 CD31, CD63 CD33, CD63 CD20, CD70CD22, CD70 CD25, CD70 CD31, CD70 CD33, CD70 CD20, CD82 CD22, CD82 CD25,CD82 CD31, CD82 CD33, CD82 CD20, CD85D CD22, CD85D CD25, CD85D CD31,CD85D CD33, CD85D CD20, CD96 CD22, CD96 CD25, CD96 CD31, CD96 CD33, CD96CD20, CD97 CD22, CD97 CD25, CD97 CD31, CD97 CD33, CD97 CD20, CD99 CD22,CD99 CD25, CD99 CD31, CD99 CD33, CD99 CD20, CD100 CD22, CD100 CD25,CD100 CD31, CD100 CD33, CD100 CD20, CD102 CD22, CD102 CD25, CD102 CD31,CD102 CD33, CD102 CD20, CD117 CD22, CD117 CD25, CD117 CD31, CD117 CD33,CD117 CD20, CD120B CD22, CD120B CD25, CD120B CD31, CD120B CD33, CD120BCD20, CD123 CD22, CD123 CD25, CD123 CD31, CD123 CD33, CD123 CD20, CD127CD22, CD127 CD25, CD127 CD31, CD127 CD33, CD127 CD20, CD133 CD22, CD133CD25, CD133 CD31, CD133 CD33, CD133 CD20, CD135 CD22, CD135 CD25, CD135CD31, CD135 CD33, CD135 CD20, CD148 CD22, CD148 CD25, CD148 CD31, CD148CD33, CD148 CD20, CD157 CD22, CD157 CD25, CD157 CD31, CD157 CD33, CD157CD20, CD172b CD22, CD172b CD25, CD172b CD31, CD172b CD33, CD172b CD20,CD191 CD22, CD191 CD25, CD191 CD31, CD191 CD33, CD191 CD20, CD217 CD22,CD217 CD25, CD217 CD31, CD217 CD33, CD217 CD20, CD300a CD22, CD300aCD25, CD300a CD31, CD300a CD33, CD300a CD20, CD305 CD22, CD305 CD25,CD305 CD31, CD305 CD33, CD305 CD20, CD317 CD22, CD317 CD25, CD317 CD31,CD317 CD33, CD317 CD20, CD321 CD22, CD321 CD25, CD321 CD31, CD321 CD33,CD321 CD20, CLL1 CD22, CLL1 CD25, CLL1 CD31, CLL1 CD33, CLL1 CD20, FOLR2CD22, FOLR2 CD25, FOLR2 CD31, FOLR2 CD33, FOLR2 CD20, NKG2D CD22, NKG2DCD25, NKG2D CD31, NKG2D CD33, NKG2D Ligand Ligand Ligand Ligand LigandCD20, EMR2 CD22, EMR2 CD25, EMR2 CD31, EMR2 CD33, EMR2 CD34, CD7 CD37,CD7 CD38, CD7 CD44, CD7 CD45, CD7 CD34, CD11a CD37, CD11a CD38, CD11aCD44, CD11a CD45, CD11a CD34, CD15 CD37, CD15 CD38, CD15 CD44, CD15CD45, CD15 CD34, CD18 CD37, CD18 CD38, CD18 CD44, CD18 CD45, CD18 CD34,CD19 CD37, CD19 CD38, CD19 CD44, CD19 CD45, CD19 CD34, CD20 CD37, CD20CD38, CD20 CD44, CD20 CD45, CD20 CD34, CD22 CD37, CD22 CD38, CD22 CD44,CD22 CD45, CD22 CD34, CD25 CD37, CD25 CD38, CD25 CD44, CD25 CD45, CD25CD34, CD31 CD37, CD31 CD38, CD31 CD44, CD31 CD45, CD31 CD34, CD33 CD37,CD33 CD38, CD33 CD44, CD33 CD45, CD33 — CD37, CD34 CD38, CD34 CD44, CD34CD45, CD34 CD34, CD37 — CD38, CD37 CD44, CD37 CD45, CD37 CD34, CD38CD37, CD38 — CD44, CD38 CD45, CD38 CD34, CD44 CD37, CD44 CD38, CD44 —CD45, CD44 CD34, CD45 CD37, CD45 CD38, CD45 CD44, CD45 — CD34, CD47CD37, CD47 CD38, CD47 CD44, CD47 CD45, CD47 CD34, CD51 CD37, CD51 CD38,CD51 CD44, CD51 CD45, CD51 CD34, CD56 CD37, CD56 CD38, CD56 CD44, CD56CD45, CD56 CD34, CD58 CD37, CD58 CD38, CD58 CD44, CD58 CD45, CD58 CD34,CD59 CD37, CD59 CD38, CD59 CD44, CD59 CD45, CD59 CD34, CD63 CD37, CD63CD38, CD63 CD44, CD63 CD45, CD63 CD34, CD70 CD37, CD70 CD38, CD70 CD44,CD70 CD45, CD70 CD34, CD82 CD37, CD82 CD38, CD82 CD44, CD82 CD45, CD82CD34, CD85D CD37, CD85D CD38, CD85D CD44, CD85D CD45, CD85D CD34, CD96CD37, CD96 CD38, CD96 CD44, CD96 CD45, CD96 CD34, CD97 CD37, CD97 CD38,CD97 CD44, CD97 CD45, CD97 CD34, CD99 CD37, CD99 CD38, CD99 CD44, CD99CD45, CD99 CD34, CD100 CD37, CD100 CD38, CD100 CD44, CD100 CD45, CD100CD34, CD102 CD37, CD102 CD38, CD102 CD44, CD102 CD45, CD102 CD34, CD117CD37, CD117 CD38, CD117 CD44, CD117 CD45, CD117 CD34, CD120B CD37,CD120B CD38, CD120B CD44, CD120B CD45, CD120B CD34, CD123 CD37, CD123CD38, CD123 CD44, CD123 CD45, CD123 CD34, CD127 CD37, CD127 CD38, CD127CD44, CD127 CD45, CD127 CD34, CD133 CD37, CD133 CD38, CD133 CD44, CD133CD45, CD133 CD34, CD135 CD37, CD135 CD38, CD135 CD44, CD135 CD45, CD135CD34, CD148 CD37, CD148 CD38, CD148 CD44, CD148 CD45, CD148 CD34, CD157CD37, CD157 CD38, CD157 CD44, CD157 CD45, CD157 CD34, CD172b CD37,CD172b CD38, CD172b CD44, CD172b CD45, CD172b CD34, CD191 CD37, CD191CD38, CD191 CD44, CD191 CD45, CD191 CD34, CD217 CD37, CD217 CD38, CD217CD44, CD217 CD45, CD217 CD34, CD300a CD37, CD300a CD38, CD300a CD44,CD300a CD45, CD300a CD34, CD305 CD37, CD305 CD38, CD305 CD44, CD305CD45, CD305 CD34, CD317 CD37, CD317 CD38, CD317 CD44, CD317 CD45, CD317CD34, CD321 CD37, CD321 CD38, CD321 CD44, CD321 CD45, CD321 CD34, CLL1CD37, CLL1 CD38, CLL1 CD44, CLL1 CD45, CLL1 CD34, FOLR2 CD37, FOLR2CD38, FOLR2 CD44, FOLR2 CD45, FOLR2 CD34, NKG2D CD37, NKG2D CD38, NKG2DCD44, NKG2D CD45, NKG2D Ligand Ligand Ligand Ligand Ligand CD34, EMR2CD37, EMR2 CD38, EMR2 CD44, EMR2 CD45, EMR2 CD47, CD7 CD51, CD7 CD56,CD7 CD58, CD7 CD59, CD7 CD47, CD11a CD51, CD11a CD56, CD11a CD58, CD11aCD59, CD11a CD47, CD15 CD51, CD15 CD56, CD15 CD58, CD15 CD59, CD15 CD47,CD18 CD51, CD18 CD56, CD18 CD58, CD18 CD59, CD18 CD47, CD19 CD51, CD19CD56, CD19 CD58, CD19 CD59, CD19 CD47, CD20 CD51, CD20 CD56, CD20 CD58,CD20 CD59, CD20 CD47, CD22 CD51, CD22 CD56, CD22 CD58, CD22 CD59, CD22CD47, CD25 CD51, CD25 CD56, CD25 CD58, CD25 CD59, CD25 CD47, CD31 CD51,CD31 CD56, CD31 CD58, CD31 CD59, CD31 CD47, CD33 CD51, CD33 CD56, CD33CD58, CD33 CD59, CD33 CD47, CD34 CD51, CD34 CD56, CD34 CD58, CD34 CD59,CD34 CD47, CD37 CD51, CD37 CD56, CD37 CD58, CD37 CD59, CD37 CD47, CD38CD51, CD38 CD56, CD38 CD58, CD38 CD59, CD38 CD47, CD44 CD51, CD44 CD56,CD44 CD58, CD44 CD59, CD44 CD47, CD45 CD51, CD45 CD56, CD45 CD58, CD45CD59, CD45 — CD51, CD47 CD56, CD47 CD58, CD47 CD59, CD47 CD47, CD51 —CD56, CD51 CD58, CD51 CD59, CD51 CD47, CD56 CD51, CD56 — CD58, CD56CD59, CD56 CD47, CD58 CD51, CD58 CD56, CD58 — CD59, CD58 CD47, CD59CD51, CD59 CD56, CD59 CD58, CD59 — CD47, CD63 CD51, CD63 CD56, CD63CD58, CD63 CD59, CD63 CD47, CD70 CD51, CD70 CD56, CD70 CD58, CD70 CD59,CD70 CD47, CD82 CD51, CD82 CD56, CD82 CD58, CD82 CD59, CD82 CD47, CD85DCD51, CD85D CD56, CD85D CD58, CD85D CD59, CD85D CD47, CD96 CD51, CD96CD56, CD96 CD58, CD96 CD59, CD96 CD47, CD97 CD51, CD97 CD56, CD97 CD58,CD97 CD59, CD97 CD47, CD99 CD51, CD99 CD56, CD99 CD58, CD99 CD59, CD99CD47, CD100 CD51, CD100 CD56, CD100 CD58, CD100 CD59, CD100 CD47, CD102CD51, CD102 CD56, CD102 CD58, CD102 CD59, CD102 CD47, CD117 CD51, CD117CD56, CD117 CD58, CD117 CD59, CD117 CD47, CD120B CD51, CD120B CD56,CD120B CD58, CD120B CD59, CD120B CD47, CD123 CD51, CD123 CD56, CD123CD58, CD123 CD59, CD123 CD47, CD127 CD51, CD127 CD56, CD127 CD58, CD127CD59, CD127 CD47, CD133 CD51, CD133 CD56, CD133 CD58, CD133 CD59, CD133CD47, CD135 CD51, CD135 CD56, CD135 CD58, CD135 CD59, CD135 CD47, CD148CD51, CD148 CD56, CD148 CD58, CD148 CD59, CD148 CD47, CD157 CD51, CD157CD56, CD157 CD58, CD157 CD59, CD157 CD47, CD172b CD51, CD172b CD56,CD172b CD58, CD172b CD59, CD172b CD47, CD191 CD51, CD191 CD56, CD191CD58, CD191 CD59, CD191 CD47, CD217 CD51, CD217 CD56, CD217 CD58, CD217CD59, CD217 CD47, CD300a CD51, CD300a CD56, CD300a CD58, CD300a CD59,CD300a CD47, CD305 CD51, CD305 CD56, CD305 CD58, CD305 CD59, CD305 CD47,CD317 CD51, CD317 CD56, CD317 CD58, CD317 CD59, CD317 CD47, CD321 CD51,CD321 CD56, CD321 CD58, CD321 CD59, CD321 CD47, CLL1 CD51, CLL1 CD56,CLL1 CD58, CLL1 CD59, CLL1 CD47, FOLR2 CD51, FOLR2 CD56, FOLR2 CD58,FOLR2 CD59, FOLR2 CD47, NKG2D CD51, NKG2D CD56, NKG2D CD58, NKG2D CD59,NKG2D Ligand Ligand Ligand Ligand Ligand CD47, EMR2 CD51, EMR2 CD56,EMR2 CD58, EMR2 CD59, EMR2 CD63, CD7 CD70, CD7 CD82, CD7 CD85D, CD7CD96, CD7 CD63, CD11a CD70, CD11a CD82, CD11a CD85D, CD11a CD96, CD11aCD63, CD15 CD70, CD15 CD82, CD15 CD85D, CD15 CD96, CD15 CD63, CD18 CD70,CD18 CD82, CD18 CD85D, CD18 CD96, CD18 CD63, CD19 CD70, CD19 CD82, CD19CD85D, CD19 CD96, CD19 CD63, CD20 CD70, CD20 CD82, CD20 CD85D, CD20CD96, CD20 CD63, CD22 CD70, CD22 CD82, CD22 CD85D, CD22 CD96, CD22 CD63,CD25 CD70, CD25 CD82, CD25 CD85D, CD25 CD96, CD25 CD63, CD31 CD70, CD31CD82, CD31 CD85D, CD31 CD96, CD31 CD63, CD33 CD70, CD33 CD82, CD33CD85D, CD33 CD96, CD33 CD63, CD34 CD70, CD34 CD82, CD34 CD85D, CD34CD96, CD34 CD63, CD37 CD70, CD37 CD82, CD37 CD85D, CD37 CD96, CD37 CD63,CD38 CD70, CD38 CD82, CD38 CD85D, CD38 CD96, CD38 CD63, CD44 CD70, CD44CD82, CD44 CD85D, CD44 CD96, CD44 CD63, CD45 CD70, CD45 CD82, CD45CD85D, CD45 CD96, CD45 CD63, CD47 CD70, CD47 CD82, CD47 CD85D, CD47CD96, CD47 CD63, CD51 CD70, CD51 CD82, CD51 CD85D, CD51 CD96, CD51 CD63,CD56 CD70, CD56 CD82, CD56 CD85D, CD56 CD96, CD56 CD63, CD58 CD70, CD58CD82, CD58 CD85D, CD58 CD96, CD58 CD63, CD59 CD70, CD59 CD82, CD59CD85D, CD59 CD96, CD59 — CD70, CD63 CD82, CD63 CD85D, CD63 CD96, CD63CD63, CD70 — CD82, CD70 CD85D, CD70 CD96, CD70 CD63, CD82 CD70, CD82 —CD85D, CD82 CD96, CD82 CD63, CD85D CD70, CD85D CD82, CD85D — CD96, CD85DCD63, CD96 CD70, CD96 CD82, CD96 CD85D, CD96 — CD63, CD97 CD70, CD97CD82, CD97 CD85D, CD97 CD96, CD97 CD63, CD99 CD70, CD99 CD82, CD99CD85D, CD99 CD96, CD99 CD63, CD100 CD70, CD100 CD82, CD100 CD85D, CD100CD96, CD100 CD63, CD102 CD70, CD102 CD82, CD102 CD85D, CD102 CD96, CD102CD63, CD117 CD70, CD117 CD82, CD117 CD85D, CD117 CD96, CD117 CD63,CD120B CD70, CD120B CD82, CD120B CD85D, CD120B CD96, CD120B CD63, CD123CD70, CD123 CD82, CD123 CD85D, CD123 CD96, CD123 CD63, CD127 CD70, CD127CD82, CD127 CD85D, CD127 CD96, CD127 CD63, CD133 CD70, CD133 CD82, CD133CD85D, CD133 CD96, CD133 CD63, CD135 CD70, CD135 CD82, CD135 CD85D,CD135 CD96, CD135 CD63, CD148 CD70, CD148 CD82, CD148 CD85D, CD148 CD96,CD148 CD63, CD157 CD70, CD157 CD82, CD157 CD85D, CD157 CD96, CD157 CD63,CD172b CD70, CD172b CD82, CD172b CD85D, CD172b CD96, CD172b CD63, CD191CD70, CD191 CD82, CD191 CD85D, CD191 CD96, CD191 CD63, CD217 CD70, CD217CD82, CD217 CD85D, CD217 CD96, CD217 CD63, CD300a CD70, CD300a CD82,CD300a CD85D, CD300a CD96, CD300a CD63, CD305 CD70, CD305 CD82, CD305CD85D, CD305 CD96, CD305 CD63, CD317 CD70, CD317 CD82, CD317 CD85D,CD317 CD96, CD317 CD63, CD321 CD70, CD321 CD82, CD321 CD85D, CD321 CD96,CD321 CD63, CLL1 CD70, CLL1 CD82, CLL1 CD85D, CLL1 CD96, CLL1 CD63,FOLR2 CD70, FOLR2 CD82, FOLR2 CD85D, FOLR2 CD96, FOLR2 CD63, NKG2D CD70,NKG2D CD82, NKG2D CD85D, NKG2D CD96, NKG2D Ligand Ligand Ligand LigandLigand CD63, EMR2 CD70, EMR2 CD82, EMR2 CD85D, EMR2 CD96, EMR2 CD97, CD7CD99, CD7 CD100, CD7 CD102, CD7 CD117, CD7 CD97, CD11a CD99, CD11aCD100, CD11a CD102, CD11a CD117, CD11a CD97, CD15 CD99, CD15 CD100, CD15CD102, CD15 CD117, CD15 CD97, CD18 CD99, CD18 CD100, CD18 CD102, CD18CD117, CD18 CD97, CD19 CD99, CD19 CD100, CD19 CD102, CD19 CD117, CD19CD97, CD20 CD99, CD20 CD100, CD20 CD102, CD20 CD117, CD20 CD97, CD22CD99, CD22 CD100, CD22 CD102, CD22 CD117, CD22 CD97, CD25 CD99, CD25CD100, CD25 CD102, CD25 CD117, CD25 CD97, CD31 CD99, CD31 CD100, CD31CD102, CD31 CD117, CD31 CD97, CD33 CD99, CD33 CD100, CD33 CD102, CD33CD117, CD33 CD97, CD34 CD99, CD34 CD100, CD34 CD102, CD34 CD117, CD34CD97, CD37 CD99, CD37 CD100, CD37 CD102, CD37 CD117, CD37 CD97, CD38CD99, CD38 CD100, CD38 CD102, CD38 CD117, CD38 CD97, CD44 CD99, CD44CD100, CD44 CD102, CD44 CD117, CD44 CD97, CD45 CD99, CD45 CD100, CD45CD102, CD45 CD117, CD45 CD97, CD47 CD99, CD47 CD100, CD47 CD102, CD47CD117, CD47 CD97, CD51 CD99, CD51 CD100, CD51 CD102, CD51 CD117, CD51CD97, CD56 CD99, CD56 CD100, CD56 CD102, CD56 CD117, CD56 CD97, CD58CD99, CD58 CD100, CD58 CD102, CD58 CD117, CD58 CD97, CD59 CD99, CD59CD100, CD59 CD102, CD59 CD117, CD59 CD97, CD63 CD99, CD63 CD100, CD63CD102, CD63 CD117, CD63 CD97, CD70 CD99, CD70 CD100, CD70 CD102, CD70CD117, CD70 CD97, CD82 CD99, CD82 CD100, CD82 CD102, CD82 CD117, CD82CD97, CD85D CD99, CD85D CD100, CD85D CD102, CD85D CD117, CD85D CD97,CD96 CD99, CD96 CD100, CD96 CD102, CD96 CD117, CD96 — CD99, CD97 CD100,CD97 CD102, CD97 CD117, CD97 CD97, CD99 — CD100, CD99 CD102, CD99 CD117,CD99 CD97, CD100 CD99, CD100 — CD102, CD100 CD117, CD100 CD97, CD102CD99, CD102 CD100, CD102 — CD117, CD102 CD97, CD117 CD99, CD117 CD100,CD117 CD102, CD117 — CD97, CD120B CD99, CD120B CD100, CD120B CD102,CD120B CD117, CD120B CD97, CD123 CD99, CD123 CD100, CD123 CD102, CD123CD117, CD123 CD97, CD127 CD99, CD127 CD100, CD127 CD102, CD127 CD117,CD127 CD97, CD133 CD99, CD133 CD100, CD133 CD102, CD133 CD117, CD133CD97, CD135 CD99, CD135 CD100, CD135 CD102, CD135 CD117, CD135 CD97,CD148 CD99, CD148 CD100, CD148 CD102, CD148 CD117, CD148 CD97, CD157CD99, CD157 CD100, CD157 CD102, CD157 CD117, CD157 CD97, CD172b CD99,CD172b CD100, CD172b CD102, CD172b CD117, CD172b CD97, CD191 CD99, CD191CD100, CD191 CD102, CD191 CD117, CD191 CD97, CD217 CD99, CD217 CD100,CD217 CD102, CD217 CD117, CD217 CD97, CD300a CD99, CD300a CD100, CD300aCD102, CD300a CD117, CD300a CD97, CD305 CD99, CD305 CD100, CD305 CD102,CD305 CD117, CD305 CD97, CD317 CD99, CD317 CD100, CD317 CD102, CD317CD117, CD317 CD97, CD321 CD99, CD321 CD100, CD321 CD102, CD321 CD117,CD321 CD97, CLL1 CD99, CLL1 CD100, CLL1 CD102, CLL1 CD117, CLL1 CD97,FOLR2 CD99, FOLR2 CD100, FOLR2 CD102, FOLR2 CD117, FOLR2 CD97, NKG2DCD99, NKG2D CD100, NKG2D CD102, NKG2D CD117, NKG2D Ligand Ligand LigandLigand Ligand CD97, EMR2 CD99, EMR2 CD100, EMR2 CD102, EMR2 CD117, EMR2CD120B, CD7 CD123, CD7 CD127, CD7 CD133, CD7 CD135, CD7 CD120B, CD11aCD123, CD11a CD127, CD11a CD133, CD11a CD135, CD11a CD120B, CD15 CD123,CD15 CD127, CD15 CD133, CD15 CD135, CD15 CD120B, CD18 CD123, CD18 CD127,CD18 CD133, CD18 CD135, CD18 CD120B, CD19 CD123, CD19 CD127, CD19 CD133,CD19 CD135, CD19 CD120B, CD20 CD123, CD20 CD127, CD20 CD133, CD20 CD135,CD20 CD120B, CD22 CD123, CD22 CD127, CD22 CD133, CD22 CD135, CD22CD120B, CD25 CD123, CD25 CD127, CD25 CD133, CD25 CD135, CD25 CD120B,CD31 CD123, CD31 CD127, CD31 CD133, CD31 CD135, CD31 CD120B, CD33 CD123,CD33 CD127, CD33 CD133, CD33 CD135, CD33 CD120B, CD34 CD123, CD34 CD127,CD34 CD133, CD34 CD135, CD34 CD120B, CD37 CD123, CD37 CD127, CD37 CD133,CD37 CD135, CD37 CD120B, CD38 CD123, CD38 CD127, CD38 CD133, CD38 CD135,CD38 CD120B, CD44 CD123, CD44 CD127, CD44 CD133, CD44 CD135, CD44CD120B, CD45 CD123, CD45 CD127, CD45 CD133, CD45 CD135, CD45 CD120B,CD47 CD123, CD47 CD127, CD47 CD133, CD47 CD135, CD47 CD120B, CD51 CD123,CD51 CD127, CD51 CD133, CD51 CD135, CD51 CD120B, CD56 CD123, CD56 CD127,CD56 CD133, CD56 CD135, CD56 CD120B, CD58 CD123, CD58 CD127, CD58 CD133,CD58 CD135, CD58 CD120B, CD59 CD123, CD59 CD127, CD59 CD133, CD59 CD135,CD59 CD120B, CD63 CD123, CD63 CD127, CD63 CD133, CD63 CD135, CD63CD120B, CD70 CD123, CD70 CD127, CD70 CD133, CD70 CD135, CD70 CD120B,CD82 CD123, CD82 CD127, CD82 CD133, CD82 CD135, CD82 CD120B, CD85DCD123, CD85D CD127, CD85D CD133, CD85D CD135, CD85D CD120B, CD96 CD123,CD96 CD127, CD96 CD133, CD96 CD135, CD96 CD120B, CD97 CD123, CD97 CD127,CD97 CD133, CD97 CD135, CD97 CD120B, CD99 CD123, CD99 CD127, CD99 CD133,CD99 CD135, CD99 CD120B, CD100 CD123, CD100 CD127, CD100 CD133, CD100CD135, CD100 CD120B, CD102 CD123, CD102 CD127, CD102 CD133, CD102 CD135,CD102 CD120B, CD117 CD123, CD117 CD127, CD117 CD133, CD117 CD135, CD117— CD123, CD120B CD127, CD120B CD133, CD120B CD135, CD120B CD120B, CD123— CD127, CD123 CD133, CD123 CD135, CD123 CD120B, CD127 CD123, CD127 —CD133, CD127 CD135, CD127 CD120B, CD133 CD123, CD133 CD127, CD133 —CD135, CD133 CD120B, CD135 CD123, CD135 CD127, CD135 CD133, CD135 —CD120B, CD148 CD123, CD148 CD127, CD148 CD133, CD148 CD135, CD148CD120B, CD157 CD123, CD157 CD127, CD157 CD133, CD157 CD135, CD157CD120B, CD172b CD123, CD172b CD127, CD172b CD133, CD172b CD135, CD172bCD120B, CD191 CD123, CD191 CD127, CD191 CD133, CD191 CD135, CD191CD120B, CD217 CD123, CD217 CD127, CD217 CD133, CD217 CD135, CD217CD120B, CD300a CD123, CD300a CD127, CD300a CD133, CD300a CD135, CD300aCD120B, CD305 CD123, CD305 CD127, CD305 CD133, CD305 CD135, CD305CD120B, CD317 CD123, CD317 CD127, CD317 CD133, CD317 CD135, CD317CD120B, CD321 CD123, CD321 CD127, CD321 CD133, CD321 CD135, CD321CD120B, CLL1 CD123, CLL1 CD127, CLL1 CD133, CLL1 CD135, CLL1 CD120B,FOLR2 CD123, FOLR2 CD127, FOLR2 CD133, FOLR2 CD135, FOLR2 CD120B, NKG2DCD123, NKG2D CD127, NKG2D CD133, NKG2D CD135, NKG2D Ligand Ligand LigandLigand Ligand CD120B, EMR2 CD123, EMR2 CD127, EMR2 CD133, EMR2 CD135,EMR2 CD148, CD7 CD157, CD7 CD172b, CD7 CD191, CD7 CD217, CD7 CD148,CD11a CD157, CD11a CD172b, CD11a CD191, CD11a CD217, CD11a CD148, CD15CD157, CD15 CD172b, CD15 CD191, CD15 CD217, CD15 CD148, CD18 CD157, CD18CD172b, CD18 CD191, CD18 CD217, CD18 CD148, CD19 CD157, CD19 CD172b,CD19 CD191, CD19 CD217, CD19 CD148, CD20 CD157, CD20 CD172b, CD20 CD191,CD20 CD217, CD20 CD148, CD22 CD157, CD22 CD172b, CD22 CD191, CD22 CD217,CD22 CD148, CD25 CD157, CD25 CD172b, CD25 CD191, CD25 CD217, CD25 CD148,CD31 CD157, CD31 CD172b, CD31 CD191, CD31 CD217, CD31 CD148, CD33 CD157,CD33 CD172b, CD33 CD191, CD33 CD217, CD33 CD148, CD34 CD157, CD34CD172b, CD34 CD191, CD34 CD217, CD34 CD148, CD37 CD157, CD37 CD172b,CD37 CD191, CD37 CD217, CD37 CD148, CD38 CD157, CD38 CD172b, CD38 CD191,CD38 CD217, CD38 CD148, CD44 CD157, CD44 CD172b, CD44 CD191, CD44 CD217,CD44 CD148, CD45 CD157, CD45 CD172b, CD45 CD191, CD45 CD217, CD45 CD148,CD47 CD157, CD47 CD172b, CD47 CD191, CD47 CD217, CD47 CD148, CD51 CD157,CD51 CD172b, CD51 CD191, CD51 CD217, CD51 CD148, CD56 CD157, CD56CD172b, CD56 CD191, CD56 CD217, CD56 CD148, CD58 CD157, CD58 CD172b,CD58 CD191, CD58 CD217, CD58 CD148, CD59 CD157, CD59 CD172b, CD59 CD191,CD59 CD217, CD59 CD148, CD63 CD157, CD63 CD172b, CD63 CD191, CD63 CD217,CD63 CD148, CD70 CD157, CD70 CD172b, CD70 CD191, CD70 CD217, CD70 CD148,CD82 CD157, CD82 CD172b, CD82 CD191, CD82 CD217, CD82 CD148, CD85DCD157, CD85D CD172b, CD85D CD191, CD85D CD217, CD85D CD148, CD96 CD157,CD96 CD172b, CD96 CD191, CD96 CD217, CD96 CD148, CD97 CD157, CD97CD172b, CD97 CD191, CD97 CD217, CD97 CD148, CD99 CD157, CD99 CD172b,CD99 CD191, CD99 CD217, CD99 CD148, CD100 CD157, CD100 CD172b, CD100CD191, CD100 CD217, CD100 CD148, CD102 CD157, CD102 CD172b, CD102 CD191,CD102 CD217, CD102 CD148, CD117 CD157, CD117 CD172b, CD117 CD191, CD117CD217, CD117 CD148, CD120B CD157, CD120B CD172b, CD120B CD191, CD120BCD217, CD120B CD148, CD123 CD157, CD123 CD172b, CD123 CD191, CD123CD217, CD123 CD148, CD127 CD157, CD127 CD172b, CD127 CD191, CD127 CD217,CD127 CD148, CD133 CD157, CD133 CD172b, CD133 CD191, CD133 CD217, CD133CD148, CD135 CD157, CD135 CD172b, CD135 CD191, CD135 CD217, CD135 —CD157, CD148 CD172b, CD148 CD191, CD148 CD217, CD148 CD148, CD157 —CD172b, CD157 CD191, CD157 CD217, CD157 CD148, CD172b CD157, CD172b —CD191, CD172b CD217, CD172b CD148, CD191 CD157, CD191 CD172b, CD191 —CD217, CD191 CD148, CD217 CD157, CD217 CD172b, CD217 CD191, CD217 —CD148, CD300a CD157, CD300a CD172b, CD300a CD191, CD300a CD217, CD300aCD148, CD305 CD157, CD305 CD172b, CD305 CD191, CD305 CD217, CD305 CD148,CD317 CD157, CD317 CD172b, CD317 CD191, CD317 CD217, CD317 CD148, CD321CD157, CD321 CD172b, CD321 CD191, CD321 CD217, CD321 CD148, CLL1 CD157,CLL1 CD172b, CLL1 CD191, CLL1 CD217, CLL1 CD148, FOLR2 CD157, FOLR2CD172b, FOLR2 CD191, FOLR2 CD217, FOLR2 CD148, NKG2D CD157, NKG2DCD172b, NKG2D CD191, NKG2D CD217, NKG2D Ligand Ligand Ligand LigandLigand CD148, EMR2 CD157, EMR2 CD172b, EMR2 CD191, EMR2 CD217, EMR2

(i) Mutated Lineage-Specific Cell-Surface Antigens

In some embodiments, the hematopoietic cells (HSCs) described herein maycontain an edited gene encoding one or more lineage-specificcell-surface proteins of interest in mutated form (mutants or variants,which are used herein interchangeably), which has reduced binding or nobinding to a cytotoxic agent as described herein. The variants may lackthe epitope to which the cytotoxic agent binds. Alternatively, themutants may carry one or more mutations of the epitope to which thecytotoxic agent binds, such that binding to the cytotoxic agent isreduced or abolished as compared to the natural or wild-typelineage-specific cell-surface protein counterpart. Such a variant ispreferred to maintain substantially similar biological activity as thewild-type counterpart.

As used herein, the term “reduced binding” refers to binding that isreduced by at least 25%. The level of binding may refer to the amount ofbinding of the cytotoxic agent to a hematopoietic cell or descendantthereof or the amount of binding of the cytotoxic agent to thelineage-specific cell-surface protein. The level of binding of ahematopoietic cell or descendant thereof that has been manipulated to acytotoxic agent may be relative to the level of binding of the cytotoxicagent to a hematopoietic cell or descendant thereof that has not beenmanipulated as determined by the same assay under the same conditions.Alternatively, the level of binding of a lineage-specific cell-surfaceprotein that lacks an epitope to a cytotoxic agent may be relative tothe level of binding of the cytotoxic agent to a lineage-specificcell-surface protein that contains the epitope (e.g., a wild-typeprotein) as determined by the same assay under the same conditions. Insome embodiments, the binding is reduced by at least 25%, 30%, 40%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In someembodiments, the binding is reduced such that there is substantially nodetectable binding in a conventional assay.

As used herein, “no binding” refers to substantially no binding, e.g.,no detectable binding or only baseline binding as determined in aconventional binding assay. In some embodiments, there is no bindingbetween the hematopoietic cells or descendants thereof that have beenmanipulated and the cytotoxic agent. In some embodiments, there is nodetectable binding between the hematopoietic cells or descendantsthereof that have been manipulated and the cytotoxic agent. In someembodiments, no binding of the hematopoietic cells or descendant thereofto the cytotoxic agent refers to a baseline level of binding, as shownusing any conventional binding assay known in the art. In someembodiments, the level of binding of the hematopoietic cells ordescendants thereof that have been manipulated and the cytotoxic agentis not biologically significant. The term “no binding” is not intendedto require the absolute absence of binding.

A cell that is “negative” for a given lineage-specific cell-surfaceantigen has a substantially reduced expression level of thelineage-specific antigen as compared with its naturally-occurringcounterpart (e.g., otherwise similar, unmodified cells), e.g., notdetectable or not distinguishable from background levels, e.g., using aflow cytometry assay, e.g., an assay of Example 1. In some instances, acell that is negative for the lineage-specific cell-surface antigen hasa level of less than 10%, 5%, 2%, or 1% of as compared with itsnaturally-occurring counterpart. The variant may share a sequencehomology of at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or above)as the wild-type counterpart and, in some embodiments, may contain noother mutations in addition to those for mutating or deleting theepitope of interest. The “percent identity” of two amino acid sequencesis determined using the algorithm of Karlin and Altschul Proc. Natl.Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and AltschulProc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm isincorporated into the NBLAST™ and XBLAST™ programs (version 2.0) ofAltschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST™ protein searchescan be performed with the XBLAST™ program, score=50, wordlength=3 toobtain amino acid sequences homologous to the protein molecules of theinvention. Where gaps exist between two sequences, Gapped BLAST™ can beutilized as described in Altschul et al., Nucleic Acids Res.25(17):3389-3402, 1997. When utilizing BLAST™ and Gapped BLAST™programs, the default parameters of the respective programs (e.g.,XBLAST™ and NBLAST™) can be used.

In some instances, the variant contains one or more amino acid residuesubstitutions (e.g., 2, 3, 4, 5, or more) within the epitope of interestsuch that the cytotoxic agent does not bind or has reduced binding tothe mutated epitope. Such a variant may have substantially reducedbinding affinity to the cytotoxic agent (e.g., having a binding affinitythat is at least 40%, 50%, 60%, 70%, 80% or 90% lower than its wild-typecounterpart). In some examples, such a variant may have abolishedbinding activity to the cytotoxic agent. In other instances, the variantcontains a deletion of a region that comprises the epitope of interest.Such a region may be encoded by an exon. In some embodiments, the regionis a domain of the lineage-specific cell-surface protein of interestthat encodes the epitope. In one example, the variant has just theepitope deleted. The length of the deleted region may range from 3-60amino acids, e.g., 5-50, 5-40, 10-30, 10-20, etc.

The mutation(s) or deletions in a mutant of a lineage-specificcell-surface antigen may be within or surround a non-essential epitopesuch that the mutation(s) or deletion(s) do not substantially affect thebioactivity of the protein.

As used herein, the term “epitope” refers to an amino acid sequence(linear or conformational) of a protein, such as a lineage-specificcell-surface antigens, that is bound by the CDRs of an antibody. In someembodiments, the cytotoxic agent binds to one or more (e.g., at least 2,3, 4, 5 or more) epitopes of a lineage-specific cell-surface antigens.In some embodiments, the cytotoxic agent binds to more than one epitopeof the lineage-specific cell-surface antigen and the hematopoietic cellsare manipulated such that each of the epitopes is absent and/orunavailable for binding by the cytotoxic agent.

In some embodiments, the genetically engineered HSCs described hereinhave one or more edited genes of lineage-specific cell-surface antigenssuch that the edited genes express mutated lineage-specific cell-surfaceantigens with mutations in one or more non-essential epitopes. Anon-essential epitope (or a fragment comprising such) refers to a domainwithin the lineage-specific protein, the mutation in which (e.g.,deletion) is less likely to substantially affect the bioactivity of thelineage-specific protein and thus the bioactivity of the cellsexpressing such. For example, when hematopoietic cells comprising adeletion or mutation of a non-essential epitope of a lineage-specificcell-surface antigen, such hematopoietic cells are able to proliferateand/or undergo erythropoietic differentiation to a similar level ashematopoietic cells that express a wild-type lineage-specificcell-surface antigen.

Non-essential epitopes of a lineage-specific cell-surface antigen can beidentified by the methods described herein or by conventional methodsrelating to protein structure-function prediction. For example, anon-essential epitope of a protein can be predicted based on comparingthe amino acid sequence of a protein from one species with the sequenceof the protein from other species. Non-conserved domains are usually notessential to the functionality of the protein. As will be evident to oneof ordinary skill in the art, non-essential epitope of a protein ispredicted using an algorithm or software, such as the PROVEAN software(see, e.g., see: provean.jcvi.org; Choi et al. PLoS ONE (2012) 7(10):e46688), to predict potential non-essential epitopes in alineage-specific protein of interest (“candidate non-essentialepitope”). Mutations, including substitution and/or deletion, many bemade in any one or more amino acid residues of a candidate non-essentialepitope using convention nucleic acid modification technologies. Theprotein variants thus prepared may be introduced into a suitable type ofcells, such as hematopoietic cells, and the functionality of the proteinvariant can be investigated to confirm that the candidate non-essentialepitope is indeed a non-essential epitope.

Alternatively, a non-essential epitope of a lineage-specificcell-surface antigen may be identified by introducing a mutation into acandidate region in a lineage-specific protein of interest in a suitabletype of host cells (e.g., hematopoietic cells) and examining thefunctionality of the mutated lineage-specific protein in the host cells.If the mutated lineage-specific protein maintains substantially thebiological activity of the native counterpart, this indicates that theregion where the mutation is introduced is non-essential to the functionof the lineage-specific protein.

Methods for assessing the functionality of the lineage-specificcell-surface antigen and the hematopoietic cells or descendants thereofwill be known in the art and include, for example, proliferation assays,differentiation assays, colony formation, expression analysis (e.g.,gene and/or protein), protein localization, intracellular signaling,functional assays, and in vivo humanized mouse models.

Any of the methods for identifying and/or verifying non-essentialepitopes in lineage-specific cell-surface antigens is also within thescope of the present disclosure.

(ii) Hematopoietic Stem Cells

In some embodiments, the hematopoietic cells described herein arehematopoietic stem cells. Hematopoietic stem cells (HSCs) are capable ofgiving rise to both myeloid and lymphoid progenitor cells that furthergive rise to myeloid cells (e.g., monocytes, macrophages, neutrophils,basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphoidcells (e.g., T cells, B cells, NK cells), respectively. HSCs arecharacterized by the expression of the cell surface marker CD34 (e.g.,CD34⁺), which can be used for the identification and/or isolation ofHSCs, and absence of cell surface markers associated with commitment toa cell lineage.

In some embodiments, the HSCs are obtained from a subject, such as amammalian subject. In some embodiments, the mammalian subject is anon-human primate, a rodent (e.g., mouse or rat), a bovine, a porcine,an equine, or a domestic animal. In some embodiments, the HSCs areobtained from a human patient, such as a human patient having ahematopoietic malignancy. In some embodiments, the HSCs are obtainedfrom a healthy donor. In some embodiments, the HSCs are obtained fromthe subject to whom the immune cells expressing the chimeric receptorswill be subsequently administered. HSCs that are administered to thesame subject from which the cells were obtained are referred to asautologous cells, whereas HSCs that are obtained from a subject who isnot the subject to whom the cells will be administered are referred toas allogeneic cells.

In some embodiments, the HSCs that are administered to the subject areallogeneic cells. In some embodiments, the HSCs are obtained from adonor having a HLA haplotype that is matched with the HLA haplotype ofthe subject. Human Leukocyte Antigen (HLA) encodes majorhistocompatibility complex (MHC) proteins in humans. MHC molecules arepresent on the surface of antigen-presenting cells as well as many othercell types and present peptides of self and non-self (e.g., foreign)antigens for immunosurveillance. However, HLA are highly polymorphic,which results in many distinct alleles. Different (foreign, non-self)alleles may be antigenic and stimulate robust adverse immune responses,particularly in organ and cell transplantation. HLA molecules that arerecognized as foreign (non-self) can result in transplant rejection. Insome embodiments, it is desirable to administer HSCs from donor that hasthe same HLA type as the patient to reduce the incidence of rejection.

The HLA loci of a donor subject may be typed to identify an individualas a HLA-matched donor for the subject. Methods for typing the HLA lociwill be evident to one of ordinary skill in the art and include, forexample, serology (serotyping), cellular typing, gene sequencing,phenotyping, and PCR methods. A HLA from a donor is considered “matched”with the HLA of the subject if the HLA loci of the donor and the subjectare identical or sufficiently similar such that an adverse immuneresponse is not expected.

In some embodiments, the HLA from the donor is not matched with the HLAof the subject. In some embodiments, the subject is administered HSCsthat are not HLA matched with the HLA of the subject. In someembodiments, the subject is further administered one or moreimmunosuppressive agents to reduce or prevent rejection of the donor HSCcells. In some embodiments, the HSCs do not comprise a CART.

HSCs may be obtained from any suitable source using convention meansknown in the art. In some embodiments, HSCs are obtained from a samplefrom a subject (or donor), such as bone marrow sample or from a bloodsample. Alternatively or in addition, HSCs may be obtained from anumbilical cord. In some embodiments, the HSCs are from bone marrow, cordblood cells, or peripheral blood mononuclear cells (PBMCs). In general,bone marrow cells may be obtained from iliac crest, femora, tibiae,spine, rib or other medullary spaces of a subject (or donor). Bonemarrow may be taken out of the patient and isolated through variousseparations and washing procedures known in the art. An exemplaryprocedure for isolation of bone marrow cells comprises the followingsteps: a) extraction of a bone marrow sample; b) centrifugal separationof bone marrow suspension in three fractions and collecting theintermediate fraction, or buffycoat; c) the buffycoat fraction from step(b) is centrifuged one more time in a separation fluid, commonlyFicoll™, and an intermediate fraction which contains the bone marrowcells is collected; and d) washing of the collected fraction from step(c) for recovery of re-transfusable bone marrow cells.

HSCs typically reside in the bone marrow but can be mobilized into thecirculating blood by administering a mobilizing agent in order toharvest HSCs from the peripheral blood. In some embodiments, the subject(or donor) from which the HSCs are obtained is administered a mobilizingagent, such as granulocyte colony-stimulating factor (G-CSF). The numberof the HSCs collected following mobilization using a mobilizing agent istypically greater than the number of cells obtained without use of amobilizing agent.

The HSCs for use in the methods described herein may express thelineage-specific cell-surface antigen of interest. Upon any of themodifications described herein (e.g., genetic modification or incubationwith a blocking agent), the HSCs would not be targeted by thecytotoxicity agent also described herein. Alternatively, the HSCs foruse in the methods described herein may not express the lineage-specificcell surface protein of interest (e.g., CD19); however, descendant cellsdifferentiated from the HSCs (e.g., B cells) express thelineage-specific cell surface protein. Upon genetic modification, anendogenous gene of the HSCs coding for the lineage-specific cell surfaceprotein may be disrupted at a region encoding a non-essential epitope ofthe lineage-specific cell surface protein. Descendant cellsdifferentiated from such modified HSCs (e.g., in vivo) would express amodified lineage-specific cell surface protein having the non-essentialepitope mutated such that they would not be targeted by the cytotoxicityagent capable of binding the non-essential epitope.

In some embodiments, a sample is obtained from a subject (or donor) andis then enriched for a desired cell type (e.g. CD34⁺/CD33⁻ cells). Forexample, PBMCs and/or CD34⁺ hematopoietic cells can be isolated fromblood as described herein. Cells can also be isolated from other cells,for example by isolation and/or activation with an antibody binding toan epitope on the cell surface of the desired cell type. Another methodthat can be used includes negative selection using antibodies to cellsurface markers to selectively enrich for a specific cell type withoutactivating the cell by receptor engagement.

Populations of HSC can be expanded prior to or after manipulating theHSC such that they don't bind the cytotoxic agent or have reducedbinding to the cytotoxic agent. The cells may be cultured underconditions that comprise an expansion medium comprising one or morecytokines, such as stem cell factor (SCF), Flt-3 ligand (Flt3L),thrombopoietin (TPO), Interleukin 3 (IL-3), or Interleukin 6 (IL-6). Thecell may be expanded for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 days or any rangenecessary. In some embodiments, the HSC are expanded after isolation ofa desired cell population (e.g., CD34⁺/CD33⁻) from a sample obtainedfrom a subject (or donor) and prior to manipulation (e.g., geneticengineering, contact with a blocking agent). In some embodiments, theHSC are expanded after genetic engineering, thereby selectivelyexpanding cells that have undergone the genetic modification and lackthe epitope (e.g., have a deletion or substitution of at least a portionof the epitope) of the lineage-specific cell-surface antigen to whichthe cytotoxic agent binds. In some embodiments, a cell (“a clone”) orseveral cells having a desired characteristic (e.g., phenotype orgenotype) following genetic modification may be selected andindependently expanded. In some embodiments, the HSC are expanded priorto contacting the HSC with a blocking agent that binds the epitope ofthe lineage-specific cell-surface antigens, thereby providing apopulation of HSC expressing the lineage-specific cell-surface antigensthat cannot be bound by the cytotoxic agent due to blocking of thecorresponding epitope by the blocking agent.

(iii) Preparation of Genetically Engineered Hematopoietic Cells

Any of the genetically engineering hematopoietic cells, such as HSCs,that carry edited genes of one or more lineage-specific cell-surfaceantigens can be prepared by a routine method or by a method describedherein. In some embodiments, the genetic engineering is performed usinggenome editing. As used herein, “genome editing” refers to a method ofmodifying the genome, including any protein-coding or non-codingnucleotide sequence, of an organism to knock out the expression of atarget gene. In general, genome editing methods involve use of anendonuclease that is capable of cleaving the nucleic acid of the genome,for example at a targeted nucleotide sequence. Repair of thedouble-stranded breaks in the genome may be repaired introducingmutations and/or exogenous nucleic acid may be inserted into thetargeted site.

Genome editing methods are generally classified based on the type ofendonuclease that is involved in generating double stranded breaks inthe target nucleic acid. These methods include use of zinc fingernucleases (ZFN), transcription activator-like effector-based nuclease(TALEN), meganucleases, and CRISPR/Cas systems.

In some embodiments, the modified cells are manipulated as describedherein using the TALEN technology known in the art. In general, TALENsare engineered restriction enzymes that can specifically bind and cleavea desired target DNA molecule. A TALEN typically contains aTranscriptional Activator-Like Effector (TALE) DNA-binding domain fusedto a DNA cleavage domain. The DNA binding domain may contain a highlyconserved 33-34 amino acid sequence with a divergent 2 amino acid RVD(repeat variable dipeptide motif) at positions 12 and 13. The RVD motifdetermines binding specificity to a nucleic acid sequence and can beengineered according to methods known to those of skill in the art tospecifically bind a desired DNA sequence. In one example, the DNAcleavage domain may be derived from the FokI endonuclease. The FokIdomain functions as a dimer, requiring two constructs with unique DNAbinding domains for sites in the target genome with proper orientationand spacing. TALENs specific to sequences in a target gene of interest(e.g., CD19, CD33) can be constructed using any method known in the art.

A TALEN specific to a target gene of interest can be used inside a cellto produce a double-stranded break (DSB). A mutation can be introducedat the break site if the repair mechanisms improperly repair the breakvia non-homologous end joining. For example, improper repair mayintroduce a frame shift mutation. Alternatively, a foreign DNA moleculehaving a desired sequence can be introduced into the cell along with theTALEN. Depending on the sequence of the foreign DNA and chromosomalsequence, this process can be used to correct a defect or introduce aDNA fragment into a target gene of interest, or introduce such a defectinto the endogenous gene, thus decreasing expression of the target gene.

In some embodiments, one or more population of hematopoietic cells isgenerated by genetic engineering of a lineage-specific cell-surfaceantigen (e.g., those described herein) using a TALEN. The geneticallyengineered hematopoietic cells may not express the lineage-specificcell-surface antigen. Alternatively, the hematopoietic cells may beengineered to express an altered version of the lineage-specificcell-surface antigen, e.g., having a deletion or mutation relative tothe wild-type counterpart. Such a mutated lineage-specific cell-surfaceantigen may preserve a certain level of the bioactivity as the wild-typecounterpart. In some embodiments, a population of hematopoietic cellscontaining a mutated CD33 is generated by genetic engineering using aTALEN. In some embodiments, exon 2 or exon 3 of CD33 is mutated using aTALEN. In some examples, a population of hematopoietic cells containinga mutated CD19 is generated by genetic engineering using a TALEN. Insome embodiments, exon 2 or exon 4 of CD19 is mutated using a TALEN.

In some embodiments, the cells can be genetically manipulated using zincfinger (ZFN) technology known in the art. In general, zinc fingermediated genomic editing involves use of a zinc finger nuclease, whichtypically comprises a DNA binding domain (i.e., zinc finger) and acleavage domain (i.e., nuclease). The zinc finger binding domain may beengineered to recognize and bind to any target gene of interest (e.g.,CD19, CD33) using methods known in the art and in particular, may bedesigned to recognize a DNA sequence ranging from about 3 nucleotides toabout 21 nucleotides in length, or from about 8 to about 19 nucleotidesin length. Zinc finger binding domains typically comprise at least threezinc finger recognition regions (e.g., zinc fingers).

Restriction endonucleases (restriction enzymes) capable ofsequence-specific binding to DNA (at a recognition site) and cleavingDNA at or near the site of binding are known in the art and may be usedto form ZFN for use in genomic editing. For example, Type IISrestriction endonucleases cleave DNA at sites removed from therecognition site and have separable binding and cleavage domains. In oneexample, the DNA cleavage domain may be derived from the FokIendonuclease.

In some embodiments, one or more population of hematopoietic cells isgenerated by genetic engineering of a lineage-specific cell-surfaceantigen (e.g., those described herein) using a ZFN. The geneticallyengineered hematopoietic cells may not express the lineage-specificcell-surface antigen. Alternatively, the hematopoietic cells may beengineered to express an altered version of the lineage-specificcell-surface antigen, e.g., having a deletion or mutation relative tothe wild-type counterpart. Such a mutated lineage-specific cell-surfaceantigen may preserve a certain level of the bioactivity as the wild-typecounterpart. In some examples, a population of hematopoietic cellscontaining a mutated CD33 is generated by genetic engineering using aZFN. In some embodiments, exon 2 or exon 3 of CD33 is mutated using aZFN. In some examples, a population of hematopoietic cells containing amutated CD19 is generated by genetic engineering using a ZFN. In someembodiments, exon 2 or exon 4 of CD19 is mutated using a ZFN.

In one aspect of the present disclosure, the replacement of cancer cellsby a modified population of normal cells is performed using normal cellsthat have been manipulated such that the cells do not bind the cytotoxicagent. Such modification may include the deletion or mutation of anepitope of the lineage specific protein using a CRISPR-Cas system, wherethe Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)-Cas system is an engineered, non-naturally occurring CRISPR-Cassystem.

The present disclosure utilizes the CRISPR/Cas system that hybridizeswith a target sequence in a lineage specific protein polynucleotide,where the CRISPR/Cas system comprises a Cas endonuclease and anengineered crRNA/tracrRNA (or single guide RNA). In some embodiments,the CRISPR/Cas system includes a crRNA and does not include a tracrRNAsequence. CRISPR/Cas complex can bind to the lineage specific proteinpolynucleotide and allow the cleavage of the protein polynucleotide,thereby modifying the polynucleotide.

The CRISPR/Cas system of the present disclosure may bind to and/orcleave the region of interest within a cell-surface lineage-specificprotein in a coding or non-coding region, within or adjacent to thegene, such as, for example, a leader sequence, trailer sequence orintron, or within a non-transcribed region, either upstream ordownstream of the coding region. The guide RNAs (gRNAs) used in thepresent disclosure may be designed such that the gRNA directs binding ofthe Cas enzyme-gRNA complexes to a pre-determined cleavage sites (targetsite) in a genome. The cleavage sites may be chosen so as to release afragment that contains a region of unknown sequence, or a regioncontaining a SNP, nucleotide insertion, nucleotide deletion,rearrangement, etc.

Cleavage of a gene region may comprise cleaving one or two strands atthe location of the target sequence by the Cas enzyme. In oneembodiment, such, cleavage can result in decreased transcription of atarget gene. In another embodiment, the cleavage can further compriserepairing the cleaved target polynucleotide by homologous recombinationwith an exogenous template polynucleotide, wherein the repair results inan insertion, deletion, or substitution of one or more nucleotides ofthe target polynucleotide.

The terms “gRNA,” “guide RNA” and “CRISPR guide sequence” may be usedinterchangeably throughout and refer to a nucleic acid comprising asequence that determines the specificity of a Cas DNA binding protein ofa CRISPR/Cas system. A gRNA hybridizes to (complementary to, partiallyor completely) a target nucleic acid sequence in the genome of a hostcell. The gRNA or portion thereof that hybridizes to the target nucleicacid may be between 15-25 nucleotides, 18-22 nucleotides, or 19-21nucleotides in length. In some embodiments, the gRNA sequence thathybridizes to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 nucleotides in length. In some embodiments, the gRNAsequence that hybridizes to the target nucleic acid is between 10-30, orbetween 15-25, nucleotides in length.

In addition to a sequence that binds to a target nucleic acid, in someembodiments, the gRNA also comprises a scaffold sequence. Expression ofa gRNA encoding both a sequence complementary to a target nucleic acidand scaffold sequence has the dual function of both binding(hybridizing) to the target nucleic acid and recruiting the endonucleaseto the target nucleic acid, which may result in site-specific CRISPRactivity. In some embodiments, such a chimeric gRNA may be referred toas a single guide RNA (sgRNA).

As used herein, a “scaffold sequence,” also referred to as a tracrRNA,refers to a nucleic acid sequence that recruits a Cas endonuclease to atarget nucleic acid bound (hybridized) to a complementary gRNA sequence.Any scaffold sequence that comprises at least one stem loop structureand recruits an endonuclease may be used in the genetic elements andvectors described herein. Exemplary scaffold sequences will be evidentto one of skill in the art and can be found, for example, in Jinek, etal. Science (2012) 337(6096):816-821, Ran, et al. Nature Protocols(2013) 8:2281-2308, PCT Application No. WO2014/093694, and PCTApplication No. WO2013/176772. In some embodiments, the CRISPR-Cassystem does not include a tracrRNA sequence.

In some embodiments, the gRNA sequence does not comprise a scaffoldsequence and a scaffold sequence is expressed as a separate transcript.In such embodiments, the gRNA sequence further comprises an additionalsequence that is complementary to a portion of the scaffold sequence andfunctions to bind (hybridize) the scaffold sequence and recruit theendonuclease to the target nucleic acid.

In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100%complementary to a target nucleic acid (see also U.S. Pat. No.8,697,359, which is incorporated by reference for its teaching ofcomplementarity of a gRNA sequence with a target polynucleotidesequence). It has been demonstrated that mismatches between a CRISPRguide sequence and the target nucleic acid near the 3′ end of the targetnucleic acid may abolish nuclease cleavage activity (Upadhyay, et al.Genes Genome Genetics (2013) 3(12):2233-2238). In some embodiments, thegRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or at least 100% complementary to the 3′ end ofthe target nucleic acid (e.g., the last 5, 6, 7, 8, 9, or 10 nucleotidesof the 3′ end of the target nucleic acid).

Example sgRNA sequences targeting intron 1, intron 2 or intron 4 of CD19are provided in Table 3. Example sgRNA sequence targeting introns 1 and2 of CD33 are provided in Table 4. Additional guide RNAs for editingCD19 and CD33 are provide below. As will be evident to one of ordinaryskill in the art, selection of sgRNA sequences may depend on factorssuch as the number of predicted on-target and/or off-target bindingsites. In some embodiments, the sgRNA sequence is selected to maximizepotential on-target and minimize potential off-target sites.

As would be evident to one of ordinary skill in the art, various toolsmay be used to design and/or optimize the sequence of a sgRNA, forexample to increase the specificity and/or precision of genomic editing.In general, candidate sgRNAs may be designed by identifying a sequencewithin the target region that has a high predicted On-target efficiencyand low Off-target efficiency based on any of the available web-basedtools. Candidate sgRNAs may be further assessed by manual inspectionand/or experimental screening. Examples of web-based tools include,without limitation, CRISPR seek, CRISPR Design Tool, Cas-OFFinder,E-CRISP, ChopChop, CasOT, CRISPR direct, CRISPOR, BREAKING-CAS,CrispRGold, and CCTop. See, e.g., Safari, et al. Current Pharma.Biotechol. (2017) 18(13).

In some embodiments, the Cas endonuclease is a Cas9 nuclease (or variantthereof) or a Cpf1 nuclease (or variant thereof). Cas9 endonucleasescleave double stranded DNA of a target nucleic acid resulting in bluntends, whereas cleavage with Cpf1 nucleases results in staggered ends ofthe nucleic acid.

In general, the target nucleic acid is flanked on the 3′ side or 5′ sideby a protospacer adjacent motif (PAM) that may interact with theendonuclease and be further involved in targeting the endonucleaseactivity to the target nucleic acid. It is generally thought that thePAM sequence flanking the target nucleic acid depends on theendonuclease and the source from which the endonuclease is derived. Forexample, for Cas9 endonucleases that are derived from Streptococcuspyogenes, the PAM sequence is NGG, although the PAM sequences NAG andNGA may be recognized with lower efficiency. For Cas9 endonucleasesderived from Staphylococcus aureus, the PAM sequence is NNGRRT (SEQ IDNO: 104). For Cas9 endonucleases that are derived from Neisseriameningitidis, the PAM sequence is NNNNGATT (SEQ ID NO: 105) or thedegenerate PAM sequence NNNNGHTT (SEQ ID NO: 106). See, e.g., AdliNature Communications (2018)9:1191. Cas9 endonucleases derived fromStreptococcus thermophilus, St1Cas9 and St3Cas9, the PAM sequences areNNAGAAW (SEQ ID NO: 107) and NGGNG (SEQ ID NO: 108), respectively. ForCas9 endonuclease derived from Treponema denticola, the PAM sequence isNAAAAC (SEQ ID NO: 109). For Cas9 endonuclease derived fromStreptococcus canis the PAM sequence is NNG (SEQ ID NO: 110). See,Chatterjee, et al. Sci. Adv. (2018) 4: eaau0766. For Cas9 endonucleasederived from Campylobacter jejuni, the PAM sequence is NNNNACAC (SEQ IDNO: 111). See, e.g., Adli Nature Communications (2018)9:1191.

In some embodiments, the Cas endonuclease is a Cpf1 nuclease. Incontrast to Cas9 endonucleases, Cpf1 endonuclease generally do notrequire a tracrRNA sequence and recognize a PAM sequence located at the5′ end of the target nucleic acid. For a Cpf1 nuclease, the PAM sequenceis TTTN (SEQ ID NO: 112). In some embodiments, the Cas endonuclease isMAD7 (also referred to as Cpf1 nuclease from Eubacterium rectale) andthe PAM sequence is YTTTN (SEQ ID NO: 113).

In some embodiments, genetically engineering a cell also comprisesintroducing a Cas endonuclease into the cell. In some embodiments, theCas endonuclease and the nucleic acid encoding the gRNA are provided onthe same nucleic acid (e.g., a vector). In some embodiments, the Casendonuclease and the nucleic acid encoding the gRNA are provided ondifferent nucleic acids (e.g., different vectors). Alternatively or inaddition, the Cas endonuclease may be provided or introduced into thecell in protein form.

In some embodiments, the Cas endonuclease is a Cas9 enzyme or variantthereof. In some embodiments, the Cas9 endonuclease is derived fromStreptococcus pyogenes (SpCas9), Staphylococcus aureus (SaCas9),Streptococcus canis (ScCas9), Neisseria meningitidis (NmCas9),Streptococcus thermophilus, Campylobacter jujuni (CjCas9), or Treponemadenticola. In some embodiments, the nucleotide sequence encoding the Casendonuclease may be codon optimized for expression in a host cell. Insome embodiments, the endonuclease is a Cas9 homolog or ortholog.

In some embodiments, the nucleotide sequence encoding the Cas9endonuclease is further modified to alter the activity of the protein.In some embodiments, the Cas9 endonuclease has been modified toinactivate one or more catalytic residues of the endonuclease. In someembodiments, the Cas9 endonuclease has been modified to inactivate oneof the catalytic residues of the endonuclease, referred to as a“nickase” or “Cas9n”. Cas9 nickase endonucleases cleave one DNA strandof the target nucleic acid. See, e.g., Dabrowska et al. Frontiers inNeuroscience (2018) 12(75). It has been shown that one or more mutationsin the RuvC and HNH catalytic domains of the enzyme may improve Cas9efficiency. See, e.g., Sarai et al. Currently Pharma. Biotechnol. (2017)18(13). In some embodiments, the Cas9 nickase comprises a mutation atamino acid position D10 and/or H840. In some examples, the Cas9 nickasecomprises the substitution mutation D10A and/or H840A.

In some embodiments, the methods described herein involve two distinctcleavage reactions, in which one Cas9 nickase is directed to cleave oneDNA strand of the target nucleic acid and a Cas9 nickase is directed tocleave the second DNA strand of the target nucleic acid.

In some embodiments, the Cas9 endonuclease is a catalytically inactiveCas9. For example, dCas9 contains mutations of catalytically activeresidues (D10 and H840) and does not have nuclease activity.Alternatively or in addition, the Cas9 endonuclease may be fused toanother protein or portion thereof. In some embodiments, dCas9 is fusedto a repressor domain, such as a KRAB domain. In some embodiments, suchdCas9 fusion proteins are used with the constructs described herein formultiplexed gene repression (e.g., CRISPR interference (CRISPRi)). Insome embodiments, dCas9 is fused to an activator domain, such as VP64 orVPR. In some embodiments, such dCas9 fusion proteins are used with theconstructs described herein for gene activation (e.g., CRISPR activation(CRISPRa)). In some embodiments, dCas9 is fused to an epigeneticmodulating domain, such as a histone demethylase domain or a histoneacetyltransferase domain. In some embodiments, dCas9 is fused to a LSD1or p300, or a portion thereof. In some embodiments, the dCas9 fusion isused for CRISPR-based epigenetic modulation. In some embodiments, dCas9or Cas9 is fused to a FokI nuclease domain (referred to as“FokI-dCas9”). In some embodiments, Cas9 or dCas9 fused to a FokInuclease domain is used for genome editing. See, e.g., Safari et al.Current Pharma. Biotechol. (2017):18. In some embodiments, Cas9 or dCas9is fused to a fluorescent protein (e.g., GFP, RFP, mCherry, etc.). Insome embodiments, Cas9/dCas9 proteins fused to fluorescent proteins areused for labeling and/or visualization of genomic loci or identifyingcells expressing the Cas endonuclease.

In some embodiments, the Cas endonuclease is modified to enhancespecificity of the enzyme (e.g., reduce off-target effects, maintainrobust on-target cleavage). In some embodiments, the Cas endonuclease isan enhanced specificity Cas9 variant (e.g., eSPCas9). See, e.g.,Slaymaker et al. Science (2016) 351 (6268): 84-88. In some embodiments,the Cas endonuclease is a high fidelity Cas9 variant (e.g., SpCas9-HF1).See, e.g., Kleinstiver et al. Nature (2016) 529: 490-495.

Cas enzymes, such as Cas endonucleases, are known in the art and may beobtained from various sources and/or engineered/modified to modulate oneor more activities or specificities of the enzymes. In some embodiments,the Cas enzyme has been engineered/modified to recognize one or more PAMsequence. In some embodiments, the Cas enzyme has beenengineered/modified to recognize one or more PAM sequence that isdifferent than the PAM sequence the Cas enzyme recognizes withoutengineering/modification. In some embodiments, the Cas enzyme has beenengineered/modified to reduce off-target activity of the enzyme.

In some embodiments, the nucleotide sequence encoding the Casendonuclease is modified further to alter the specificity of theendonuclease activity (e.g., reduce off-target cleavage, decrease theCas endonuclease activity or lifetime in cells, increasehomology-directed recombination and reduce non-homologous end joining).See, e.g., Komor et al. Cell (2017) 168: 20-36. In some embodiments, thenucleotide sequence encoding the Cas endonuclease is modified to alterthe PAM recognition of the endonuclease. For example, the Casendonuclease SpCas9 recognizes PAM sequence NGG, whereas relaxedvariants of the SpCas9 comprising one or more modifications of theendonuclease (e.g., VQR SpCas9, EQR SpCas9, VRER SpCas9) may recognizethe PAM sequences NGA, NGAG (SEQ ID NO: 114), NGCG (SEQ ID NO: 115). PAMrecognition of a modified Cas endonuclease is considered “relaxed” ifthe Cas endonuclease recognizes more potential PAM sequences as comparedto the Cas endonuclease that has not been modified. For example, the Casendonuclease SaCas9 recognizes PAM sequence NNGRRT (SEQ ID NO: 104),whereas a relaxed variant of the SaCas9 comprising one or moremodifications of the endonuclease (e.g., KKH SaCas9) may recognize thePAM sequence NNNRRT (SEQ ID NO: 116). In one example, the Casendonuclease FnCas9 recognizes PAM sequence NNG, whereas a relaxedvariant of the FnCas9 comprising one or more modifications of theendonuclease (e.g., RHA FnCas9) may recognize the PAM sequence YG. Inone example, the Cas endonuclease is a Cpf1 endonuclease comprisingsubstitution mutations S542R and K607R and recognize the PAM sequenceTYCV. In one example, the Cas endonuclease is a Cpf1 endonucleasecomprising substitution mutations S542R, K607R, and N552R and recognizethe PAM sequence TATV. See, e.g., Gao et al. Nat. Biotechnol. (2017)35(8): 789-792.

In some embodiments, the methods described herein involve geneticallyengineering a population of hematopoietic cells using a Cas9 nuclease(or variant thereof). In some embodiments, the methods described hereininvolve genetically engineering a gene encoding a type 1lineage-specific cell-surface antigen in a population of hematopoieticcells using a Cas9 nuclease (or variant thereof). In some embodiments,the methods described herein involve genetically modifying or editing aCD19 gene, or genetically modifying or editing a CD33 gene, orgenetically modifying or editing a CD19 gene and a CD33 gene in thepopulation of hematopoietic cells using a Cas9 nuclease (or variantthereof). In some embodiments, the methods described herein involvegenetically engineering a mutant CD19 gene in a population ofhematopoietic cells using a Cas9 nuclease (or variant thereof). In someembodiments, the methods described herein involve geneticallyengineering a mutation in exon 2 or exon 4 of CD19 in a population ofhematopoietic cells using a Cas9 nuclease (or variant thereof). In someembodiments, the methods described herein involve geneticallyengineering a mutant CD19 gene in a population of hematopoietic cellsusing a Cas9 nuclease (or variant thereof) and a guide sequence providedby any one of SEQ ID NOs: 14-26, 67, and 69-72. In some embodiments, themethods described herein involve genetically engineering a mutant CD19gene in a population of hematopoietic cells using a Cas9 nuclease (orvariant thereof) and a guide sequence provided by SEQ ID NO: 67.

In some embodiments, the methods described herein involve geneticallyengineering a gene encoding a type 2 lineage-specific cell-surfaceantigen in a population of hematopoietic cells using a Cas9 nuclease (orvariant thereof). In some embodiments, the methods described hereininvolve genetically engineering a mutant CD33 gene in a population ofhematopoietic cells using a Cas9 nuclease (or variant thereof). In someembodiments, the methods described herein involve geneticallyengineering a mutation in exon 2 or exon 3 of CD33 in a population ofhematopoietic cells using a Cas9 nuclease (or variant thereof). In someembodiments, the methods described herein involve geneticallyengineering a mutant CD33 gene in a population of hematopoietic cellsusing a Cas9 nuclease (or variant thereof) and a guide sequence providedby any one of SEQ ID NOs: 27-50 and 68. In some embodiments, the methodsdescribed herein involve genetically engineering a mutant CD33 gene in apopulation of hematopoietic cells using a Cas9 nuclease (or variantthereof) and a guide sequence provided by SEQ ID NO: 68.

In some embodiments, the endonuclease is a base editor. Base editorendonuclease generally comprises a catalytically inactive Casendonuclease fused to a function domain. See, e.g., Eid et al. Biochem.J. (2018) 475(11): 1955-1964; Rees et al. Nature Reviews Genetics (2018)19:770-788. In some embodiments, the catalytically inactive Casendonuclease is dCas9. In some embodiments, the endonuclease comprises adCas9 fused to one or more uracil glycosylase inhibitor (UGI) domains.In some embodiments, the endonuclease comprises a dCas9 fused to anadenine base editor (ABE), for example an ABE evolved from the RNAadenine deaminase TadA. In some embodiments, the endonuclease comprisesa dCas9 fused to cytodine deaminase enzyme (e.g., APOBEC deaminase,pmCDA1, activation-induced cytidine deaminase (AID)). In someembodiments, the catalytically inactive Cas endonuclease has reducedactivity and is nCas9. In some embodiments, the endonuclease comprises anCas9 fused to one or more uracil glycosylase inhibitor (UGI) domains.In some embodiments, the endonuclease comprises a nCas9 fused to anadenine base editor (ABE), for example an ABE evolved from the RNAadenine deaminase TadA. In some embodiments, the endonuclease comprisesa nCas9 fused to cytodine deaminase enzyme (e.g., APOBEC deaminase,pmCDA1, activation-induced cytidine deaminase (AID)).

Examples of base editors include, without limitation, BE1, BE2, BE3,HF-BE3, BE4, BE4max, BE4-Gam, YE1-BE3, EE-BE3, YE2-BE3, YEE-CE3,VQR-BE3, VRER-BE3, SaBE3, SaBE4, SaBE4-Gam, Sa(KKH)-BE3, Target-AID,Target-AID-NG, xBE3, eA3A-BE3, BE-PLUS, TAM, CRISPR-X, ABE7.9, ABE7.10,ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, and CRISPR-SKIP.Additional examples of base editors can be found, for example, in USPublication No. 2018/0312825A1, US Publication No. 2018/0312828A1, andPCT Publication No. WO 2018/165629A1, which are incorporated byreference herein in their entireties.

In some embodiments, the base editor has been further modified toinhibit base excision repair at the target site and induce cellularmismatch repair. Any of the Cas endonucleases described herein may befused to a Gam domain (bacteriophage Mu protein) to protect the Casendonuclease from degradation and exonuclease activity. See, e.g., Eidet al. Biochem. J. (2018) 475(11): 1955-1964.

In some embodiments, the methods described herein involve geneticallyengineering a population of hematopoietic cells using a base editor (orvariant thereof). In some embodiments, the methods described hereininvolve genetically engineering a gene encoding a type 1lineage-specific cell-surface antigen in a population of hematopoieticcells using a base editor (or variant thereof). In some embodiments, themethods described herein involve genetically modifying or editing a CD19gene, or genetically modifying or editing a CD33 gene, or geneticallymodifying or editing a CD19 gene and a CD33 gene in the population ofhematopoietic cells using a base editor (or variant thereof). In someembodiments, the methods described herein involve geneticallyengineering a mutant CD19 gene in a population of hematopoietic cellsusing a base editor (or variant thereof). In some embodiments, themethods described herein involve genetically engineering a mutation inexon 2 or exon 4 of CD19 in a population of hematopoietic cells using abase editor (or variant thereof). In some embodiments, the methodsdescribed herein involve genetically engineering a mutant CD19 gene in apopulation of hematopoietic cells using a base editor (or variantthereof) and a guide sequence provided by any one of SEQ ID NOs: 14-26,67, and 69-72. In some embodiments, the methods described herein involvegenetically engineering a mutant CD19 gene in a population ofhematopoietic cells using a base editor (or variant thereof) and a guidesequence provided by SEQ ID NO: 67.

In some embodiments, the methods described herein involve geneticallyengineering a gene encoding a type 2 lineage-specific cell-surfaceantigen in a population of hematopoietic cells using a base editor (orvariant thereof). In some embodiments, the methods described hereininvolve genetically engineering a mutant CD33 gene in a population ofhematopoietic cells using a base editor (or variant thereof). In someembodiments, the methods described herein involve geneticallyengineering a mutation in exon 2 or exon 3 of CD33 in a population ofhematopoietic cells using a base editor nuclease (or variant thereof).In some embodiments, the methods described herein involve geneticallyengineering a mutant CD33 gene in a population of hematopoietic cellsusing a base editor nuclease (or variant thereof) and a guide sequenceprovided by any one of SEQ ID NOs: 27-50 and 68. In some embodiments,the methods described herein involve genetically engineering a mutantCD33 gene in a population of hematopoietic cells using a base editor (orvariant thereof) and a guide sequence provided by SEQ ID NO: 68.

In some embodiments, the Cas endonuclease belongs to class 2 type V ofCas endonuclease. Class 2 type V Cas endonucleases can be furthercategorized as type V-A, type V-B, type V-C, and type V-U. See, e.g.,Stella et al. Nature Structural & Molecular Biology (2017). In someembodiments, the Cas endonuclease is a type V-A Cas endonuclease, suchas a Cpf1 nuclease. In some embodiments, the Cas endonuclease is a typeV-B Cas endonuclease, such as a C2c1 endonuclease. See, e.g., Shmakov etal. Mol Cell (2015) 60: 385-397. In some embodiments, the Casendonuclease is Mad7.

In some embodiments, the Cas endonuclease is a Cpf1 nuclease or variantthereof. As will be appreciated by one of skill in the art, the Casendonuclease Cpf1 nuclease may also be referred to as Cas12a. See, e.g.,Strohkendl et al. Mol. Cell (2018) 71: 1-9. In some embodiments, thehost cell expresses a Cpf1 nuclease derived from Provetella spp.,Francisella spp, Acidaminococcus sp. (AsCpf1), Lachnospiraceae bacterium(LpCpf1), or Eubacterium rectale. In some embodiments, the nucleotidesequence encoding the Cpf1 nuclease may be codon optimized forexpression in a host cell. In some embodiments, the nucleotide sequenceencoding the Cpf1 endonuclease is further modified to alter the activityof the protein.

A catalytically inactive variant of Cpf1 (Cas12a) may be referred todCas12a. As described herein, catalytically inactive variants of Cpf1maybe fused to a function domain to form a base editor. See, e.g., Reeset al. Nature Reviews Genetics (2018) 19:770-788. In some embodiments,the catalytically inactive Cas endonuclease is dCas9. In someembodiments, the endonuclease comprises a dCas12a fused to one or moreuracil glycosylase inhibitor (UGI) domains. In some embodiments, theendonuclease comprises a dCas12a fused to an adenine base editor (ABE),for example an ABE evolved from the RNA adenine deaminase TadA. In someembodiments, the endonuclease comprises a dCas12a fused to cytodinedeaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-inducedcytidine deaminase (AID)).

In some embodiments, the methods described herein involve geneticallyengineering a population of hematopoietic cells using a Cpf1 nuclease(or variant thereof. In some embodiments, the methods described hereininvolve genetically engineering a gene encoding a type 1lineage-specific cell-surface antigen in a population of hematopoieticcells using a Cpf1 nuclease (or variant thereof. In some embodiments,the methods described herein involve genetically modifying or editing aCD19 gene, or genetically modifying or editing a CD33 gene, orgenetically modifying or editing a CD19 gene and a CD33 gene in thepopulation of hematopoietic cells using a Cpf1 nuclease (or variantthereof. In some embodiments, the methods described herein involvegenetically engineering a mutant CD19 gene in a population ofhematopoietic cells using a Cpf1 nuclease (or variant thereof. In someembodiments, the methods described herein involve geneticallyengineering a mutation in exon 2 or exon 4 of CD19 in a population ofhematopoietic cells using a Cpf1 nuclease (or variant thereof). In someembodiments, the methods described herein involve geneticallyengineering a mutant CD19 gene in a population of hematopoietic cellsusing a Cpf1 nuclease (or variant thereof) and a guide sequence providedby any one of SEQ ID NOs: 14-26, 67, and 69-72. In some embodiments, themethods described herein involve genetically engineering a mutant CD19gene in a population of hematopoietic cells using a Cpf1 nuclease (orvariant thereof) and a guide sequence provided by SEQ ID NO: 67.

In some embodiments, the methods described herein involve geneticallyengineering a gene encoding a type 2 lineage-specific cell-surfaceantigen in a population of hematopoietic cells using a Cpf1 nuclease (orvariant thereof). In some embodiments, the methods described hereininvolve genetically engineering a mutant CD33 gene in a population ofhematopoietic cells using a Cpf1 nuclease (or variant thereof). In someembodiments, the methods described herein involve geneticallyengineering a mutation in exon 2 or exon 3 of CD33 in a population ofhematopoietic cells using a Cpf1 nuclease (or variant thereof). In someembodiments, the methods described herein involve geneticallyengineering a mutant CD33 gene in a population of hematopoietic cellsusing a Cpf1 nuclease (or variant thereof) and a guide sequence providedby any one of SEQ ID NOs: 27-50 and 68. In some embodiments, the methodsdescribed herein involve genetically engineering a mutant CD33 gene in apopulation of hematopoietic cells using a Cpf1 nuclease (or variantthereof) and a guide sequence provided by SEQ ID NO: 68.

Alternatively or in addition, the Cas endonuclease may be a Cas14endonuclease or variant thereof. In contrast to Cas9 endonucleases,Cas14 endonucleases are derived from archaea and tend to be smaller insize (e.g., 400-700 amino acids). Additionally Cas14 endonucleases donot require a PAM sequence. See, e.g., Harrington et al. Science (2018).

In some embodiments, the methods described herein involve geneticallyengineering a population of hematopoietic cells using a Cas14endonuclease (or variant thereof. In some embodiments, the methodsdescribed herein involve genetically engineering a gene encoding a type1 lineage-specific cell-surface antigen in a population of hematopoieticcells using a Cas14 endonuclease (or variant thereof. In someembodiments, the methods described herein involve genetically modifyingor editing a CD19 gene, or genetically modifying or editing a CD33 gene,or genetically modifying or editing a CD19 gene and a CD33 gene in thepopulation of hematopoietic cells using a Cas14 endonuclease (or variantthereof). In some embodiments, the methods described herein involvegenetically engineering a mutant CD19 gene in a population ofhematopoietic cells using a Cas14 endonuclease (or variant thereof). Insome embodiments, the methods described herein involve geneticallyengineering a mutation in exon 2 or exon 4 of CD19 in a population ofhematopoietic cells using a Cas14 endonuclease (or variant thereof). Insome embodiments, the methods described herein involve geneticallyengineering a mutant CD19 gene in a population of hematopoietic cellsusing a Cas14 endonuclease (or variant thereof) and a guide sequenceprovided by any one of SEQ ID NOs: 14-26, 67, and 69-72. In someembodiments, the methods described herein involve geneticallyengineering a mutant CD19 gene in a population of hematopoietic cellsusing a Cas14 endonuclease (or variant thereof) and a guide sequenceprovided by SEQ ID NO: 67.

In some embodiments, the methods described herein involve geneticallyengineering a gene encoding a type 2 lineage-specific cell-surfaceantigen in a population of hematopoietic cells using a Cas14endonuclease (or variant thereof. In some embodiments, the methodsdescribed herein involve genetically engineering a mutant CD33 gene in apopulation of hematopoietic cells using a Cas14 endonuclease (or variantthereof). In some embodiments, the methods described herein involvegenetically engineering a mutation in exon 2 or exon 3 of CD33 in apopulation of hematopoietic cells using a Cas14 endonuclease (or variantthereof). In some embodiments, the methods described herein involvegenetically engineering a mutant CD33 gene in a population ofhematopoietic cells using a Cas14 endonuclease (or variant thereof) anda guide sequence provided by any one of SEQ ID NOs: 27-50 and 68. Insome embodiments, the methods described herein involve geneticallyengineering a mutant CD33 gene in a population of hematopoietic cellsusing a Cas14 endonuclease (or variant thereof) and a guide sequenceprovided by SEQ ID NO: 68.

Any of the Cas endonucleases described herein may be modulated toregulate levels of expression and/or activity of the Cas endonuclease ata desired time. For example, it may be advantageous to increase levelsof expression and/or activity of the Cas endonuclease during particularphase(s) of the cell cycle. It has been demonstrated that levels ofhomology-directed repair are reduced during the G1 phase of the cellcycle, therefore increasing levels of expression and/or activity of theCas endonuclease during the S phase, G2 phase, and/or M phase mayincrease homology-directed repair following the Cas endonucleaseediting. In some embodiments, levels of expression and/or activity ofthe Cas endonuclease are increased during the S phase, G2 phase, and/orM phase of the cell cycle. In one example, the Cas endonuclease fused toa the N-terminal region of human Geminin. See, e.g., Gutschner et al.Cell Rep. (2016) 14(6): 1555-1566. In some embodiments, levels ofexpression and/or activity of the Cas endonuclease are reduced duringthe G1 phase. In one example, the Cas endonuclease is modified such thatit has reduced activity during the G1 phase. See, e.g., Lomova et al.Stem Cells (2018).

Alternatively or in addition, any of the Cas endonucleases describedherein may be fused to an epigenetic modifier (e.g., achromatin-modifying enzyme, e.g., DNA methylase, histone deacetylase).See, e.g., Kungulovski et al. Trends Genet. (2016) 32(2):101-113. Casendonucleases fused to an epigenetic modifier may be referred to as“epieffectors” and may allow for temporal and/or transient endonucleaseactivity. In some embodiments, the Cas endonuclease is a dCas9 fused toa chromatin-modifying enzyme.

In some embodiments, the present disclosure provides compositions andmethods for modifying or deleting a cell-surface lineage-specificprotein in hematopoietic cells using a CRISPR/Cas9 system, wherein guideRNA sequence hybridizes to the nucleotide sequence encoding an epitopeof the lineage-specific cell-surface antigen. In some embodiments, thepresent disclosure provides compositions and methods for modifying ordeleting two or more cell-surface lineage-specific protein inhematopoietic cells using a CRISPR/Cas9 system, wherein guide RNAsequence hybridizes to the nucleotide sequence encoding an epitope ofthe lineage-specific cell-surface antigen. In some embodiments, theguide RNA sequence(s) hybridize to the nucleotide sequence encoding anexon of the lineage-specific cell-surface antigen. In some embodiments,one or more guide RNA sequences may hybridize to one or more intronsequences, leading to skipping of an adjacent exon. For example, twoguide RNA sequence may be used to target regions in two nearby introns(e.g., intron 1 and intron 2 or intron 2 and intron 3), leading toskipping of the exon between the two introns. In some embodiments, thecell-surface lineage-specific protein is CD33 or CD19 and the gRNAhybridizes to a portion of the nucleotide sequence that encodes anepitope of CD33 or CD19. In some embodiments, the cell-surfacelineage-specific protein is CD33 and the gRNA hybridizes to a portion ofintron 1 or intron 2 of the nucleotide sequence encoding CD19. In someembodiments, the cell-surface lineage-specific protein is CD19 and thegRNA hybridizes to a portion of intron 1 or intron 2 of the nucleotidesequence encoding CD19.

In some embodiments, the cell-surface lineage-specific protein is CD33and the gRNA hybridizes to a portion of the gene encoding exon2. In someembodiments, the cell-surface lineage-specific protein is CD18 and thegRNA hybridizes to a portion of the gene encoding exon3. In someembodiments, a first cell-surface lineage-specific protein is CD33(wherein the gRNA hybridizes to a portion of the gene encoding exon2 anda second cell-surface lineage-specific protein is CD19 and the gRNAhybridizes to a portion of exon3. In some embodiments, the CD33 and/orCD19 gene is knocked out. In some embodiments, a portion of the CD33and/or CD19 gene is knocked out.

In some embodiments, it may be desired to further genetically engineerthe HSC, particularly allogeneic HSCs, to reduce the graft-versus-hosteffects. For example, the standard therapy for relapsed AML ishematopoietic stem cell transplantation (HSCT). However, at least one ofthe limiting factors for successful HSCT is graft-versus-host disease(GVHD), in which expression of the cell surface molecule CD45 has beenimplicated. See, e.g., Van Besie, Hematology Am. Soc. Hematol EducProgram (2013)56; Mawad, Curr. Hematol. Malig. Rep. (2013) 8(2):132.CD45RA and CD45RO are isoforms of CD45 (found on all hematopoietic cellsexcept erythrocytes). In T lymphocytes, CD45RA is expressed on naivecells, while CD45RO is expressed on memory cells. CD45RA T cells have ahigh potential for reactivity against recipient-specific proteinsfollowing HSCT, resulting in GVHD. CD45 is a type 1 lineage protein, asCD45-bearing cells are required for survival; however, the antigenicportion of CD45 may be deleted from stem cells using CRISPR to preventand/or reduce the incidence or extent of GvHD.

Also provided herein are methods of producing the genetically engineeredhematopoietic cells as described herein, which carry edited genes forexpressing one or more lineage-specific cell-surface antigens in mutatedform. Such methods may involve providing a cell and introducing into thecell components of a CRISPR Cas system for genome editing. In someembodiments, a nucleic acid that comprises a CRISPR-Cas guide RNA (gRNA)that hybridizes or is predicted to hybridize to a portion of thenucleotide sequence that encodes the lineage-specific cell-surfaceantigen is introduced into the cell. In some embodiments, the gRNA isintroduced into the cell on a vector. In some embodiments, a Casendonuclease is introduced into the cell. In some embodiments, the Casendonuclease is introduced into the cell as a nucleic acid encoding aCas endonuclease. In some embodiments, the gRNA and a nucleotidesequence encoding a Cas endonuclease are introduced into the cell on thesame nucleic acid (e.g., the same vector). In some embodiments, the Casendonuclease is introduced into the cell in the form of a protein. Insome embodiments, the Cas endonuclease and the gRNA are pre-formed invitro and are introduced to the cell in as a ribonucleoprotein complex.

In some embodiments, multiple gRNAs are introduced into the cell. Insome embodiments, the two or more guide RNAs are transfected into cellsin equimolar amounts. In some embodiments, the two or more guide RNAsare provided in amounts that are not equimolar. In some embodiments, thetwo or more guide RNAs are provided in amounts that are optimized sothat editing of each target occurs at equal frequency. In someembodiments, the two or more guide RNAs are provided in amounts that areoptimized so that editing of each target occurs at optimal frequency.

In some embodiments, multiple gRNAs are allowed to form gRNA-RNPcomplexes in the same reaction. In some embodiments, two or moregRNA-RNP complexes are formed in separate reactions. The RNP complexeswith the two or more guide RNAs can be transfected together orseparately. For example, Cas9-CD19_gRNA-19 RNPs and Cas9-CD33_gRNA-37RNPs can be formed separately in two isolated incubations or together inone incubation and can be transfected together or separately, e.g.,concurrently.

In some embodiments, the two or more guides are transfected concurrentlywith each other. In some embodiments, the two or more guides areprovided sequentially or consecutively, i.e., in two or more separatetransfections. For example, Cas9-CD19_gRNA-RNPs and Cas9-CD33 gRNA RNPscan be transfected together, e.g., in equimolar amounts or anotheroptimal ratio. In some examples, RNPs comprising Cas9 and any one of theCD19 gRNAs provided by SEQ ID NOs: 14-26, 67, 69-72 are transfected withRNPs comprising Cas9 and any one of the CD33 gRNAs provided by SEQ IDNOs: 22-50 and 68. Alternatively, Cas9-CD19_gRNA-RNPs and Cas9-CD33 gRNARNPs can be transfected sequentially, e.g., either firstCas9-CD19_gRNA-RNPs and then Cas9-CD33 gRNA RNPs or firstCas9-CD33_gRNA-RNPs and then Cas9-CD19 gRNA RNPs. In some examples, RNPscomprising Cas9 and any one of the CD19 gRNAs provided by SEQ ID NOs:14-26, 67, 69-72 are transfected sequentially (e.g., prior to or after)RNPs comprising Cas9 and any one of the CD33 gRNAs provided by SEQ IDNOs: 22-50 and 68.

Vectors of the present disclosure can drive the expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, Nature(1987) 329: 840) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6: 187).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

The vectors of the present disclosure are capable of directingexpression of the nucleic acid preferentially in a particular cell type(e.g., tissue-specific regulatory elements are used to express thenucleic acid). Such regulatory elements include promoters that may betissue specific or cell specific. The term “tissue-specific” as itapplies to a promoter refers to a promoter that is capable of directingselective expression of a nucleotide sequence of interest to a specifictype of tissue (e.g., seeds) in the relative absence of expression ofthe same nucleotide sequence of interest in a different type of tissue.The term “cell type specific” as applied to a promoter refers to apromoter that is capable of directing selective expression of anucleotide sequence of interest in a specific type of cell in therelative absence of expression of the same nucleotide sequence ofinterest in a different type of cell within the same tissue. The term“cell type specific” when applied to a promoter also means a promotercapable of promoting selective expression of a nucleotide sequence ofinterest in a region within a single tissue. Cell type specificity of apromoter may be assessed using methods well known in the art, e.g.,immunohistochemical staining.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding CRISPR/Cas9 in mammalian cells ortarget tissues. Such methods can be used to administer nucleic acidsencoding components of a CRISPR-Cas system to cells in culture, or in ahost organism. Non-viral vector delivery systems include DNA plasmids,RNA (e.g., a transcript of a vector described herein), naked nucleicacid, and nucleic acid complexed with a delivery vehicle. In someembodiments, nucleic acids encoding CRISPR/Cas9 are introduced bytransfection (e.g., electroporation, microinjection. In someembodiments, nucleic acids encoding CRISPR/Cas9 are introduced bynanoparticle delivery, e.g., cationic nanocarriers.

Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell.

Viral vectors can be administered directly to patients (in vivo) or theycan be used to manipulate cells in vitro or ex vivo, where the modifiedcells may be administered to patients. In one embodiment, the presentdisclosure utilizes viral based systems including, but not limited toretroviral, lentivirus, adenoviral, adeno-associated and herpes simplexvirus vectors for gene transfer. Furthermore, the present disclosureprovides vectors capable of integration in the host genome, such asretrovirus or lentivirus. Preferably, the vector used for the expressionof a CRISPR-Cas system of the present disclosure is a lentiviral vector.

In one embodiment, the disclosure provides for introducing one or morevectors encoding CRISPR-Cas into eukaryotic cell. The cell can be acancer cell. Alternatively, the cell is a hematopoietic cell, such as ahematopoietic stem cell. Examples of stem cells include pluripotent,multipotent and unipotent stem cells. Examples of pluripotent stem cellsinclude embryonic stem cells, embryonic germ cells, embryonic carcinomacells and induced pluripotent stem cells (iPSCs). In a preferredembodiment, the disclosure provides introducing CRISPR-Cas9 into ahematopoietic stem cell.

The vectors of the present disclosure are delivered to the eukaryoticcell in a subject. Modification of the eukaryotic cells via CRISPR/Cas9system can takes place in a cell culture, where the method comprisesisolating the eukaryotic cell from a subject prior to the modification.In some embodiments, the method further comprises returning saideukaryotic cell and/or cells derived therefrom to the subject.

In some embodiments, the gRNA is introduced into the cell in the form ofa vector. In some embodiments, the gRNA and a nucleotide sequenceencoding a Cas endonuclease are introduced into the cell on the samenucleic acid (e.g., the same vector). In some embodiments, the gRNA isintroduced into the cell in the form of an RNA. In some embodiments, thegRNA may comprise one or more modifications, for example, to enhancestability of the gRNA, reduce off-target activity, increase editingefficiency. Examples of modifications include, without limitation, basemodifications, backbone modifications, and modifications to the lengthof the gRNA. For example, it has been demonstrated that extending thelength of a gRNA at the 5′end and/or introducing one or more chemicalmodification may increase editing efficiency. See, e.g., Park et al.Nature Communications (2018) 9:3313; Moon et al. Nature Communications(2018) 9: 3651. Additionally, incorporation of nucleic acids or lockednucleic acids have been found to increase specificity of genomicediting. See, e.g., Cromwell, et al. Nature Communications (2018) 9:1448. See, e.g., Safari et al. Current Pharm. Biotechnol. (2017) 18:13.In some embodiments, the gRNA may comprise one or more modificationchosen from phosphorothioate backbone modification, 2′-O-Me-modifiedsugars (e.g., at one or both of the 3′ and 5′ termini), 2′F-modifiedsugar, replacement of the ribose sugar with the bicyclic nucleotide-cEt,3′thioPACE (MSP), or any combination thereof. Suitable gRNAmodifications are described, e.g., in Randar et al. PNAS Dec. 22, 2015112 (51) E7110-E7117 and Hendel et al., Nat Biotechnol. 2015 September;33(9): 985-989, each of which is incorporated herein by reference in itsentirety. In some embodiments, a gRNA described herein comprises one ormore 2′-O-methyl-3′-phosphorothioate nucleotides, e.g., at least 2, 3,4, 5, or 6 2′-O-methyl-3′-phosphorothioate nucleotides. In someembodiments, a gRNA described herein comprises modified nucleotides(e.g., 2′-O-methyl-3′-phosphorothioate nucleotides) at the threeterminal positions and the 5′ end and/or at the three terminal positionsand the 3′ end.

In some embodiments, the gRNA comprises one or more modified bases (e.g.2′ O-methyl nucleotides). In some embodiments, the gRNA comprises one ormore modified uracil base. In some embodiments, the gRNA comprises oneor more modified adenine base. In some embodiments, the gRNA comprisesone or more modified guanine base. In some embodiments, the gRNAcomprises one or more modified cytosine base.

In some embodiments, the gRNA comprises one or more modifiedinternucleotide linkages such as, for example, phosphorothioate,phosphoramidate, and O′methyl ribose or deoxyribose residue.

In some embodiments, the gRNA comprises an extension of about 10nucleotides to 100 nucleotides at the 3′ end and/or 5′end of the gRNA.In some embodiments, the gRNA comprises an extension of about 10nucleotides to 100 nucleotides, about 20 nucleotides to 90 nucleotides,about 30 nucleotides to 80 nucleotides, about 40 nucleotides to 70nucleotides, about 40 nucleotides to 60 nucleotides, about 50nucleotides to 60 nucleotides.

In some embodiments, the Cas endonuclease and the gRNA are pre-formed invitro and are introduced to the cell in as a ribonucleoprotein complex.Examples of mechanisms to introduce a ribonucleoprotein complexcomprising the Cas endonuclease and the gRNA include, withoutlimitation, electroporation, cationic lipids, DNA nanoclew, and cellpenetrating peptides. See, e.g., Safari et al. Current Pharma.Biotechnol. (2017) 18(13); Yin et al. Nature Review Drug Discovery(2017) 16: 387-399.

Any of the CRISPR/Cas systems described herein may be further optimizedto increase selectivity of genomic editing, for example by enhancinghomologous recombination. See, e.g., Komor et al. Cell (2017) 168:20-36. For example, in some embodiments, CRISPR/Cas system is optimizedto inhibit nonhomologous end joining and/or promote homologous directedrecombination.

A number of small molecules have been identified to modulate Casendonuclease genome editing. In some embodiments, the cells arecontacted with one or more small molecule to enhance Cas endonucleasegenome editing. In some embodiments, a subject is administered one ormore small molecule to enhance Cas endonuclease genome editing. In someembodiments, the cells are contacted with one or more small molecule toinhibit nonhomologous end joining and/or promote homologous directedrecombination. Examples of small molecules that may modulate Casendonuclease genome editing include, without limitation L755507,Brefeldin A, ligase IV inhibitor SCR7, VE-822, AZD-7762. See, e.g., Huet al. Cell Chem. Biol. (2016) 23: 57-73; Yu et al. Cell Stem Cell(2015) 16: 142-147; Chu et al. Nat. Biotechnol. (2015) 33: 543-548;Maruyama et al. Nat. Biotechnol. (2015) 33: 538-542; and Ma et al.Nature Communications (2018) 9:1303.

In some embodiments, any of the Cas endonucleases may be used with adonor single stranded DNA designed to anneal with the DNA strandinitially released from the Cas endonucleases.

In some embodiments, it is desirable to temporally regulate genomicediting. For example, in some embodiments, the expression and/oractivity of a Cas endonuclease may be regulated to induce genomicediting at a desired time. In some embodiments, cells containing any ofthe CRISPR/Cas systems described herein may be allowed to engraft into asubject and then expression and/or activity the Cas endonuclease may beinduced. Alternatively or in addition, as described herein, the Casendonuclease may be fused to an epigenetic modifier (e.g., achromatin-modifying enzyme, e.g., DNA methylase, histone deacetylase).

(B) Genetically Engineered Hematopoietic Cells Expressing CD19 Mutantsand/or CD33 Mutants

In some embodiments, the genetically engineered hematopoietic cells mayhave edited CD19 gene, CD33 gene, or both, which are designed to expressmutated CD19, CD33, or both. In some instances, the mutated CD19 and/orCD33 include mutations or deletions in one or more non-essentialepitopes so as to retain (at least partially) the bioactivity of CD19and/or CD33.

(i) Genetically Engineered Hematopoietic Cells Expressing CD19 Mutants

In some examples, provided herein are variants of CD19, which maycomprise a deletion or mutation of a fragment of the protein that isencoded by any one of the exons of CD19, or deletion or mutation in anon-essential epitope of CD19. The whole sequence of the CD19 gene,containing fifteen exons, is known in the art. See, e.g., GenBankaccession no. NC_000016. For example, one or more epitopes located inthe region encoded by exon 2 the CD19 gene may be deleted or mutated.Certain modifications to the region of the CD19 gene encoding exon 2have been shown to result in successful CD19 protein expression,membrane localization, and partial maintenance of protein function(Sotillo et al. Cancer Discovery. (2015) 5: 1282-1295). For example,missense or frameshift mutations in exon 2 of the CD19 gene, oralternatively, modifications that permanently or transiently reduceexpression of the splicing factor SRSF3, which is involved in retentionof CD19 exon 2, may reduce CD19 expression in vivo. In some embodiments,one or more epitopes located in the region encoded by exon 2 of the CD19gene are mutated or deleted. For example, the FMC63 epitope of CD19,which is a known target of CD19-targeted CAR therapies may be mutated ordeleted (Sotillo et al. Cancer Discovery. (2015) 5: 1282-129; Nicholsonet al. Mol Immunol. (1997) 34:1157-1165; Zola et al. Immunol Cell Biol.(1991) 69:411-422).

In some examples, one or more epitopes located in the region encoded byexon 4 of the CD19 gene may be deleted or mutated.

In some embodiments, exon 2 of CD19 is mutated or deleted. In someembodiments, exon 4 of CD19 is mutated or deleted. The amino acidsequence of an exemplary human CD19 is provided below with the fragmentencoded by exon 2 underlined and the fragment encoded by exon 4 initalics (SEQ ID NO:51).

(SEQ ID NO: 51) MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR  CD19 Full-lengthmRNA (underlined = exon 2; italics = exon 4) (SEQ ID NO: 117)AUGCCACCUCCUCGCCUCCUCUUCUUCCUCCUCUUCCUCACCCCCAUGGAAGUCAGGCCCGAGGAACCUCUAGUGGUGAAGGUGGAAGAGGGAGAUAACGCUGUGCUGCAGUGCCUCAAGGGGACCUCAGAUGGCCCCACUCAGCAGCUGACCUGGUCUCGGGAGUCCCCGCUUAAACCCUUCUUAAAACUCAGCCUGGGGCUGCCAGGCCUGGGAAUCCACAUGAGGCCCCUGGCCAUCUGGCUUUUCAUCUUCAACGUCUCUCAACAGAUGGGGGGCUUCUACCUGUGCCAGCCGGGGCCCCCCUCUGAGAAGGCCUGGCAGCCUGGCUGGACAGUCAAUGUGGAGGGCAGCGGGGAGCUGUUCCGGUGGAAUGUUUCGGACCUAGGUGGCCUGGGCUGUGGCCUGAAGAACAGGUCCUCAGAGGGCCCCAGCUCCCCUUCCGGGAAGCUCAUGAGCCCCAAGCUGUAUGUGUGGGCCAAAGACCGCCCUGAGAUCUGGGAGGGAGAGCCUCCGUGUCUCCCACCGAGGGACAGCCUGAACCAGAGCCUCAGCCAGGACCUCACCAUGGCCCCUGGCUCCACACUCUGGCUGUCCUGUGGGGUACCCCCUGACUCUGUGUCCAGGGGCCCCCUCUCCUGGACCCAUGUGCACCCCAAGGGGCCUAAGUCAUUGCUGAGCCUAGAGCUGAAGGACGAUCGCCCGGCCAGAGAUAUGUGGGUAAUGGAGACGGGUCUGUUGUUGCCCCGGGCCACAGCUCAAGACGCUGGAAAGUAUUAUUGUCACCGUGGCAACCUGACCAUGUCAUUCCACCUGGAGAUCACUGCUCGGCCAGUACUAUGGCACUGGCUGCUGAGGACUGGUGGCUGGAAGGUCUCAGCUGUGACUUUGGCUUAUCUGAUCUUCUGCCUGUGUUCCCUUGUGGGCAUUCUUCAUCUUCAAAGAGCCCUGGUCCUGAGGAGGAAAAGAAAGCGAAUGACUGACCCCACCAGGAGAUUCUUCAAAGUGACGCCUCCCCCAGGAAGCGGGCCCCAGAACCAGUACGGGAACGUGCUGUCUCUCCCCACACCCACCUCAGGCCUCGGACGCGCCCAGCGUUGGGCCGCAGGCCUGGGGGGCACUGCCCCGUCUUAUGGAAACCCGAGCAGCGACGUCCAGGCGGAUGGAGCCUUGGGGUCCCGGAGCCCGCCGGGAGUGGGCCCAGAAGAAGAGGAAGGGGAGGGCUAUGAGGAACCUGACAGUGAGGAGGACUCCGAGUUCUAUGAGAACGACUCCAACCUUGGGCAGGACCAGCUCUCCCAGGAUGGCAGCGGCUACGAGAACCCUGAGGAUGAGCCCCUGGGUCCUGAGGAUGAAGACUCCUUCUCCAACGCUGAGUCUUAUGAGAACGAGGAUGAAGAGCUGACCCAGCCGGUCGCCAGGACAAUGGACUUCCUGAGCCCUCAUGGGUCAGCCUGGGACCCCAGCCGGGAAGCAACCUCCCUGGCAGGGUCCCAGUCCUAUGAGGAUAUGAGAGGAAUCCUGUAUGCAGCCCCCCAGCUCCGCUCCAUUCGGGGCCAGCCUGGACCCAAUCAUGAGGAAGAUGCAGACUCUUAUGAGAACAUGGAUAAUCCCGAUGGGCCAGACCCAGCCUGGGGAGGAGGGGGCCGCAUGGGCACCUGGAGCACCAGGUGA

In some examples, the genetically engineered hematopoietic cells have agenetically engineered CD19 gene (e.g., a genetically engineeredendogenous CD19 gene), which expresses a CD19 mutant having the fragmentencoded by exon 2 deleted (CD19ex2). An exemplary amino acid sequence ofsuch a CD19 mutant is provided below (the junction of exon 1-encodedfragment and exon-3 encoded fragment is shown in boldface):

(SEQ ID NO: 52) MPPPRLLFFLLFLTPMEVRPEEPLVVKVEGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLAGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGG GRMGTWSTRmRNA ex2 delete (SEQ ID NO: 118)AUGCCACCUCCUCGCCUCCUCUUCUUCCUCCUCUUCCUCACCCCCAUGGAAGUCAGGCCCGAGGAACCUCUAGUGGUGAAGGUGGAAGGGGAGCUGUUCCGGUGGAAUGUUUCGGACCUAGGUGGCCUGGGCUGUGGCCUGAAGAACAGGUCCUCAGAGGGCCCCAGCUCCCCUUCCGGGAAGCUCAUGAGCCCCAAGCUGUAUGUGUGGGCCAAAGACCGCCCUGAGAUCUGGGAGGGAGAGCCUCCGUGUCUCCCACCGAGGGACAGCCUGAACCAGAGCCUCAGCCAGGACCUCACCAUGGCCCCUGGCUCCACACUCUGGCUGUCCUGUGGGGUACCCCCUGACUCUGUGUCCAGGGGCCCCCUCUCCUGGACCCAUGUGCACCCCAAGGGGCCUAAGUCAUUGCUGAGCCUAGAGCUGAAGGACGAUCGCCCGGCCAGAGAUAUGUGGGUAAUGGAGACGGGUCUGUUGUUGCCCCGGGCCACAGCUCAAGACGCUGGAAAGUAUUAUUGUCACCGUGGCAACCUGACCAUGUCAUUCCACCUGGAGAUCACUGCUCGGCCAGUACUAUGGCACUGGCUGCUGAGGACUGGUGGCUGGAAGGUCUCAGCUGUGACUUUGGCUUAUCUGAUCUUCUGCCUGUGUUCCCUUGUGGGCAUUCUUCAUCUUCAAAGAGCCCUGGUCCUGAGGAGGAAAAGAAAGCGAAUGACUGACCCCACCAGGAGAUUCUUCAAAGUGACGCCUCCCCCAGGAAGCGGGCCCCAGAACCAGUACGGGAACGUGCUGUCUCUCCCCACACCCACCUCAGGCCUCGGACGCGCCCAGCGUUGGGCCGCAGGCCUGGGGGGCACUGCCCCGUCUUAUGGAAACCCGAGCAGCGACGUCCAGGCGGAUGGAGCCUUGGGGUCCCGGAGCCCGCCGGGAGUGGGCCCAGAAGAAGAGGAAGGGGAGGGCUAUGAGGAACCUGACAGUGAGGAGGACUCCGAGUUCUAUGAGAACGACUCCAACCUUGGGCAGGACCAGCUCUCCCAGGAUGGCAGCGGCUACGAGAACCCUGAGGAUGAGCCCCUGGGUCCUGAGGAUGAAGACUCCUUCUCCAACGCUGAGUCUUAUGAGAACGAGGAUGAAGAGCUGACCCAGCCGGUCGCCAGGACAAUGGACUUCCUGAGCCCUCAUGGGUCAGCCUGGGACCCCAGCCGGGAAGCAACCUCCCUGGCAGGGUCCCAGUCCUAUGAGGAUAUGAGAGGAAUCCUGUAUGCAGCCCCCCAGCUCCGCUCCAUUCGGGGCCAGCCUGGACCCAAUCAUGAGGAAGAUGCAGACUCUUAUGAGAACAUGGAUAAUCCCGAUGGGCCAGACCCAGCCUGGGGAGGAGGGGGCCGCAUGGGCACCUGGAGCACCAGGUGA

In some embodiments, exon 4 of CD19 is mutated or deleted. In someexamples, the genetically engineered hematopoietic cells have agenetically engineered CD19 gene (e.g., a genetically engineeredendogenous CD19 gene), which expresses a CD19 mutant having the fragmentencoded by exon 4 deleted (CD19ex4). An exemplary amino acid sequence ofsuch a CD19 mutant is provided below:

(SEQ ID NO: 73) MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLAGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRM GTWSTRmRNA ex4 delete (SEQ ID NO: 119)ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCCCGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGGCCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACCTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCTGTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAGCCCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGTGTCTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGTACTATGGCACTGGCTGCTGAGGACTGGTGGCTGGAAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCTTGTGGGCATTCTTCATCTTCAAAGAGCCCTGGTCCTGAGGAGGAAAAGAAAGCGAATGACTGACCCCACCAGGAGATTCTTCAAAGTGACGCCTCCCCCAGGAAGCGGGCCCCAGAACCAGTACGGGAACGTGCTGTCTCTCCCCACACCCACCTCAGGCCTCGGACGCGCCCAGCGTTGGGCCGCAGGCCTGGGGGGCACTGCCCCGTCTTATGGAAACCCGAGCAGCGACGTCCAGGCGGATGGAGCCTTGGGGTCCCGGAGCCCGCCGGGAGTGGGCCCAGAAGAAGAGGAAGGGGAGGGCTATGAGGAACCTGACAGTGAGGAGGACTCCGAGTTCTATGAGAACGACTCCAACCTTGGGCAGGACCAGCTCTCCCAGGATGGCAGCGGCTACGAGAACCCTGAGGATGAGCCCCTGGGTCCTGAGGATGAAGACTCCTTCTCCAACGCTGAGTCTTATGAGAACGAGGATGAAGAGCTGACCCAGCCGGTCGCCAGGACAATGGACTTCCTGAGCCCTCATGGGTCAGCCTGGGACCCCAGCCGGGAAGCAACCTCCCTGGCAGGGTCCCAGTCCTATGAGGATATGAGAGGAATCCTGTATGCAGCCCCCCAGCTCCGCTCCATTCGGGGCCAGCCTGGACCCAATCATGAGGAAGATGCAGACTCTTATGAGAACATGGATAATCCCGATGGGCCAGACCCAGCCTGGGGAGGAGGGGGCCGCATGGGCACCTGGAGCACCAGGTGA

Genetically engineered hematopoietic stem cells carrying an edited CD19gene that expresses this CD19 mutant are also within the scope of thepresent disclosure.

Genetically engineered hematopoietic stem cells carrying an edited CD19gene that expresses this CD19 mutant are also within the scope of thepresent disclosure. Such cells may be a homogenous population containingcells expressing the same CD19 mutant (e.g., CD19ex2, CD19ex4).Alternatively, the cells may be a heterogeneous population containingcells expressing different CD19 mutants (which may due to heterogeneousediting/repairing events inside cells) or cells that do not express CD19(CD19KO). In specific examples, the genetically engineered HSCs may be aheterogeneous population containing cells expressing CD19ex2 and cellsthat do not express CD19 (CD19KO). In some specific examples, thegenetically engineered HSCs may be a heterogeneous population containingcells expressing CD19ex4 and cells that do not express CD19 (CD19KO).

Genetically engineered hematopoietic stem cells having edited a CD19gene can be prepared by a suitable genome editing method, such as thoseknown in the art or disclosed herein. In some embodiments, thegenetically engineered hematopoietic stem cells described herein can begenerated using the CRISPR approach. See discussions herein. In certainexamples, specific guide RNAs targeting a fragment of the CD19 gene (anexon sequence or an intron sequence) can be used in the CRISPR method.Exemplary gRNAs for editing the CD19 gene (e.g., deletion of exon 2,exon 4) are provided in Example 1, Table 3, and Example 3 below.

In some examples, multiple gRNAs can be used for editing the CD19 genevia CRISPR. Different combinations of gRNAs, e.g., selected from thoselisted in Table 3, can be used in the multiplex approach. In oneexample, the pair of gRNA 6 (AGCAGAGGACTCCAAAAGCT; SEQ ID NO: 18) andgRNA 14 (CCATGGACAGAAGAGGTCCG; SEQ ID NO: 24) are used for editing CD19via CRISPR. Also provided herein are methods of genetically editing CD19in hematopoietic cells (e.g., HSCs) via CRISPR, using one or more of thegRNAs described herein, for example, the pair of gRNA6+gRNA14 or thepair of gRNA 23+gRNA 24.

Because of the mechanism of Cas9 cutting and DNA repair, there will be aspectrum of repair events including small insertions on 1-2 nucleotidesand occasionally longer deletions. Representative sequences of repairedCD19exon 2 deletion products (intron 1-intron 2 displayed) are shownbelow (SEQ ID Nos:53-55):

Example Repair Sequence Length Comments #1 Ligation:CCGGCTCCTCCACTCCCagcccgCGGCCACAATGGAGCTGGAG 0 #2 Insertion:CCGGCTCCTCCACTCCCagcTccgCGGCCACAATGGAGCTGGAG +1 #3 Deletion:---------------------------------------gCGGCCACAATGGAGCTGGAG −133Partial loss Exon 1Despite the heterogeneity at the genomic DNA level, the RNA transcriptsprovided from the edited CD19 gene all encode a CD19 mutant having thefragment encoded by exon 2 deleted.

(ii) Genetically Engineered Hematopoietic Cells Expressing CD33 Mutants

In some embodiments, the lineage-specific cell-surface protein is CD33.As will be known to one of ordinary skill in the art, CD33 is encoded byseven exons, including the alternatively spliced exons 7A and 7B(Brinkman-Van der Linden et al. Mol Cell. Biol. (2003) 23: 4199-4206).Further, the CD33 gene encodes two isoforms, one of which retains exon2, referred to as CD33M, and one that excludes exon 2, referred to asCD33m (FIG. 17).

Exemplary amino acid sequences of the 7A and 7B splicing isoforms areprovided below:

CD33M-7A: Amino Acid (underlined = exon 2; italicized = exon 7A)(SEQ ID NO: 1) MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNP SKDTSTEYSEVRTQCD33-M; transcript variant 1 mRNA. NM_001772 (underlined = exon 2;italicized = exon 7A) (SEQ ID NO: 120)AUGCCGCUGCUGCUACUGCUGCCCCUGCUGUGGGCAGGGGCCCUGGCUAUGGAUCCAAAUUUCUGGCUGCAAGUGCAGGAGUCAGUGACGGUACAGGAGGGUUUGUGCGUCCUCGUGCCCUGCACUUUCUUCCAUCCCAUACCCUACUACGACAAGAACUCCCCAGUUCAUGGUUACUGGUUCCGGGAAGGAGCCAUUAUAUCCAGGGACUCUCCAGUGGCCACAAACAAGCUAGAUCAAGAAGUACAGGAGGAGACUCAGGGCAGAUUCCGCCUCCUUGGGGAUCCCAGUAGGAACAACUGCUCCCUGAGCAUCGUAGACGCCAGGAGGAGGGAUAAUGGUUCAUACUUCUUUCGGAUGGAGAGAGGAAGUACCAAAUACAGUUACAAAUCUCCCCAGCUCUCUGUGCAUGUGACAGACUUGACCCACAGGCCCAAAAUCCUCAUCCCUGGCACUCUAGAACCCGGCCACUCCAAAAACCUGACCUGCUCUGUGUCCUGGGCCUGUGAGCAGGGAACACCCCCGAUCUUCUCCUGGUUGUCAGCUGCCCCCACCUCCCUGGGCCCCAGGACUACUCACUCCUCGGUGCUCAUAAUCACCCCACGGCCCCAGGACCACGGCACCAACCUGACCUGUCAGGUGAAGUUCGCUGGAGCUGGUGUGACUACGGAGAGAACCAUCCAGCUCAACGUCACCUAUGUUCCACAGAACCCAACAACUGGUAUCUUUCCAGGAGAUGGCUCAGGGAAACAAGAGACCAGAGCAGGAGUGGUUCAUGGGGCCAUUGGAGGAGCUGGUGUUACAGCCCUGCUCGCUCUUUGUCUCUGCCUCAUCUUCUUCAUAGUGAAGACCCACAGGAGGAAAGCAGCCAGGACAGCAGUGGGCAGGAAUGACACCCACCCUACCACAGGGUCAGCCUCCCCGAAACACCAGAAGAAGUCCAAGUUACAUGGCCCCACUGAAACCUCAAGCUGUUCAGGUGCCGCCCCUACUGUGGAGAUGGAUGAGGAGCUGCAUUAUGCUUCCCUCAACUUUCAUGGGAUGAAUCCUUCCAAGGACACCUCCACCGAAUACUCAGAGGUCAGGACCCAGUGA CD33m-7A: Amino Acid(italicized = exon 7A; SEQ ID NO: 56)MPLLLLLPLLWADLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQCD33-m transcript variant 2; no exon 2, exon 7AmRNA. NM_001082618 (italicized = exon 7A)) (SEQ ID NO: 121)AUGCCGCUGCUGCUACUGCUGCCCCUGCUGUGGGCAGACUUGACCCACAGGCCCAAAAUCCUCAUCCCUGGCACUCUAGAACCCGGCCACUCCAAAAACCUGACCUGCUCUGUGUCCUGGGCCUGUGAGCAGGGAACACCCCCGAUCUUCUCCUGGUUGUCAGCUGCCCCCACCUCCCUGGGCCCCAGGACUACUCACUCCUCGGUGCUCAUAAUCACCCCACGGCCCCAGGACCACGGCACCAACCUGACCUGUCAGGUGAAGUUCGCUGGAGCUGGUGUGACUACGGAGAGAACCAUCCAGCUCAACGUCACCUAUGUUCCACAGAACCCAACAACUGGUAUCUUUCCAGGAGAUGGCUCAGGGAAACAAGAGACCAGAGCAGGAGUGGUUCAUGGGGCCAUUGGAGGAGCUGGUGUUACAGCCCUGCUCGCUCUUUGUCUCUGCCUCAUCUUCUUCAUAGUGAAGACCCACAGGAGGAAAGCAGCCAGGACAGCAGUGGGCAGGAAUGACACCCACCCUACCACAGGGUCAGCCUCCCCGAAACACCAGAAGAAGUCCAAGUUACAUGGCCCCACUGAAACCUCAAGCUGUUCAGGUGCCGCCCCUACUGUGGAGAUGGAUGAGGAGCUGCAUUAUGCUUCCCUCAACUUUCAUGGGAUGAAUCCUUCCAAGGACACCUCCACCGAAUACUCAGAGG UCAGGACCCAGUGACD33M-7B: Amino Acid (underlined = exon 2; italicized = exon 7B;SEQ ID NO: 57) MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTH PTTGSASPVRCD33 transcript variant 3; exon 7B mRNA. NM_001177608(underlined = exon 2; italicized = exon 7B)) (SEQ ID NO: 122)AUGCCGCUGCUGCUACUGCUGCCCCUGCUGUGGGCAGGGGCCCUGGCUAUGGAUCCAAAUUUCUGGCUGCAAGUGCAGGAGUCAGUGACGGUACAGGAGGGUUUGUGCGUCCUCGUGCCCUGCACUUUCUUCCAUCCCAUACCCUACUACGACAAGAACUCCCCAGUUCAUGGUUACUGGUUCCGGGAAGGAGCCAUUAUAUCCAGGGACUCUCCAGUGGCCACAAACAAGCUAGAUCAAGAAGUACAGGAGGAGACUCAGGGCAGAUUCCGCCUCCUUGGGGAUCCCAGUAGGAACAACUGCUCCCUGAGCAUCGUAGACGCCAGGAGGAGGGAUAAUGGUUCAUACUUCUUUCGGAUGGAGAGAGGAAGUACCAAAUACAGUUACAAAUCUCCCCAGCUCUCUGUGCAUGUGACAGACUUGACCCACAGGCCCAAAAUCCUCAUCCCUGGCACUCUAGAACCCGGCCACUCCAAAAACCUGACCUGCUCUGUGUCCUGGCCUGUGAGCAGGGAACACCCCCGAUCUUCUCCGGUUGUCAGCUGCCCCCACCUCCCUGGGCCCCAGGACUACUCACUCCUCGGUGCUCAUAAUCACCCCACGGCCCCAGGACCACGGCACCAACCUGACCUGUCAGGUGAAGUUCGCUGGAGCUGGUGUGACUACGGAGAGAACCAUCCAGCUCAACGUCACCUAUGUUCCACAGAACCCAACAACUGGUAUCUUUCCGGAGAUGGCUCAGGGAAACAAGAGACCAGAGCAGGAGUGGUUCAUGGGGCCAUUGGAGGAGCUGGUGUUACAGCCCUGCUCGCUCUUUGUCUCUGCCUCAUCUUCUUCAUAGUGAAGACCCACAGGAGGAAAGCAGCCAGGACAGCAGUGGGCAGGAAUGACACCCACCCUACCACAGGGUCAGCCUCCCCGGUACGUUGA CD33m-7B Amino Acid(italicized = exon 7B; SEQ ID NO: 58)MPLLLLLPLLWADLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPVRCD33 transcript variant 4; no exon 2, exon 7B.mRNA (italicized = exon 7B) (SEQ ID NO: 123)AUGCCGCUGCUGCUACUGCUGCCCCUGCUGUGGGCAAGGAACAACUGCUCCCUGAGCAUCGUAGACGCCAGGAGGAGGGAUAAUGGUUCAUACUUCUUUCGGAUGGAGAGAGGAAGUACCAAAUACAGUUACAAAUCUCCCCAGCUCUCUGUGCAUGUGACAGACUUGACCCACAGGCCCAAAAUCCUCAUCCCUGGCACUCUAGAACCCGGCCACUCCAAAAACCUGACCUGCUCUGUGUCCUGGCCUGUGAGCAGGGAACACCCCCGAUCUUCUCCGGUUGUCAGCUGCCCCCACCUCCCUGGGCCCCAGGACUACUCACUCCUCGGUGCUCAUAAUCACCCCACGGCCCCAGGACCACGGCACCAACCUGACCUGUCAGGUGAAGUUCGCUGGAGCUGGUGUGACUACGGAGAGAACCAUCCAGCUCAACGUCACCUAUGUUCCACAGAACCCAACAACUGGUAUCUUUCCGGAGAUGGCUCAGGGAAACAAGAGACCAGAGCAGGAGUGGUUCAUGGGGCCAUUGGAGGAGCUGGUGUUACAGCCCUGCUCGCUCUUUGUCUCUGCCUCAUCUUCUUCAUAGUGAAGACCCACAGGAGGAAAGCAGCCAGGACAGCAGUGGGCAGGAAUGACACCCACCCUACCACAGGGUCAGCCUCCCCGGUACGUUGA

Using human CD33 as an exemplary lineage-specific cell-surface protein,regions of the protein in which mutation and/or deletion of amino acidsare less likely to result in deleterious effects (e.g., a reduction orabrogation of function) were predicted using PROVEAN software (see:provean.jcvi.org; Choi et al. PLoS ONE (2012) 7(10): e46688). Examplesof the predicted regions are shown in boxes in FIG. 2 and exemplarydeletions in the predicated regions are presented in Table 2. Numberingof the amino acid residues is based on the amino acid sequence of humanCD33 provided by SEQ ID NO: 1 (CD33M-7A).

TABLE 2 Exemplary deletions in CD33 Epitope targeted DeletionPROVEAN Score by cytotoxic agent S248-E252 −5.508 SGKQE (SEQ ID NO: 8)I47-D51 −5.661 IPYYD (SEQ ID NO: 9) G249-T253 −7.078GKQET (SEQ ID NO: 10) K250-R254 −7.184 KQETR (SEQ ID NO: 11) P48-K52−7.239 PYYDK (SEQ ID NO: 12) Q251-A255 −7.888 QETRA (SEQ ID NO: 13)

The nucleotide sequence encoding CD33 are genetically manipulated todelete any epitope of the protein (of the extracellular portion ofCD33), or a fragment containing such, using conventional methods ofnucleic acid manipulation. The amino acid sequences provided below areexemplary sequences of CD33 mutants that have been manipulated to lackeach of the epitopes in Table 2.

The amino acid sequence of the extracellular portion of CD33 is providedby SEQ ID NO: 1. The signal peptide is shown in italics and sites formanipulation are shown in underline and boldface. The transmembranedomain is shown in italics with underline.

(SEQ ID NO: 1) MPLLLLLPLL WAGALAMDPN FWLQVQESVT VQEGLCVLVP CTFFHPIPYY DKNS PVHGYWFREGAIISRD SPVATNKLDQ EVQEETQGRF RLLGDPSRNN CSLSIVDARR RDNGSYFFRMERGSTKYSYK SPQLSVHVTD LTHRPKILIP GTLEPGHSKN LTCSVSWACE QGTPPIFSWLSAAPTSLGPR TTHSSVLIIT PRPQDHGTNL TCQVKFAGAG VTTERTIQLN VTYVPQNPTT GIFPGDGSGK QETRAG VVH G AIGGAGVTAL LALCLCLIFF IV KTHRRKAA RTAVGRNDTHPTTGSASPKH QKKSKLHGPT ETSSCSGAAP TVEMDEELHY ASLNFHGMNP SKDTSTEYSE VRTQ

The amino acid sequence of the extracellular portion of CD33 comprisinga deletion of residues S248 through E252 is provided by SEQ ID NO: 2.The signal peptide is shown in italics and the transmembrane domain isshown in italics with underline.

S248_E252insdelTARND; PROVEAN score = −1.916 (SEQ ID NO: 2)MPLLLLLPLL WAGALAMDPN FWLQVQESVT VQEGLCVLVP CTFFHPIPYY DKNSPVHGYWFREGAIISRD SPVATNKLDQ EVQEETQGRF RLLGDPSRNN CSLSIVDARR RDNGSYFFRMERGSTKYSYK SPQLSVHVTD LTHRPKILIP GTLEPGHSKN LTCSVSWACE QGTPPIFSWLSAAPTSLGPR TTHSSVLIIT PRPQDHGTNL TCQVKFAGAG VTTERTIQLN VTYVPQNPTTGIFPGDGTAR NDTRAGVVH G   AIGGAGVTAL LALCLCLIFF IV KTHRRKAA RTAVGRNDTHPTTGSASPKH QKKSKLHGPT ETSSCSGAAP TVEMDEELHY ASLNFHGMNP SKDTSTEYSE VRTQ

The amino acid sequence of the extracellular portion of CD33 comprisinga deletion of residues I47 through D51 is provided by SEQ ID NO: 3. Thesignal peptide is shown in italics and the transmembrane domain is shownin italics with underline. I47_D51insdelVPFFE; PROVEAN score=−1.672

I47_D51insdelVPFFE; PROVEAN score = −1.672 (SEQ ID NO: 3)MPLLLLLPLL WAGALAMDPN FWLQVQESVT VQEGLCVLVP CTFFHPVPFF EKNSPVHGYWFREGAIISRD SPVATNKLDQ EVQEETQGRF RLLGDPSRNN CSLSIVDARR RDNGSYFFRMERGSTKYSYK SPQLSVHVTD LTHRPKILIP GTLEPGHSKN LTCSVSWACE QGTPPIFSWLSAAPTSLGPR TTHSSVLIIT PRPQDHGTNL TCQVKFAGAG VTTERTIQLN VTYVPQNPTTGIFPGDGTAR NDTRAGVVH G   AIGGAGVTAL   LALCLCLIFF   IVKTHRRKAA RTAVGRNDTHPTTGSASPKH QKKSKLHGPT ETSSCSGAAP TVEMDEELHY ASLNFHGMNP SKDTSTEYSE VRTQ

The amino acid sequence of the extracellular portion of CD33 comprisinga deletion of residues G249 through T253 is provided by SEQ ID NO: 4.The signal peptide is shown in italics and the transmembrane domain isshown in italics with underline.

(SEQ ID NO: 4)MPLLLLLPLL WAGALAMDPN FWLQVQESVT VQEGLCVLVP CTFFHPIPYY DKNSPVHGYWFREGAIISRD SPVATNKLDQ EVQEETQGRF RLLGDPSRNN CSLSIVDARR RDNGSYFFRMERGSTKYSYK SPQLSVHVTD LTHRPKILIP GTLEPGHSKN LTCSVSWACE QGTPPIFSWLSAAPTSLGPR TTHSSVLIIT PRPQDHGTNL TCQVKFAGAG VTTERTIQLN VTYVPQNPTTGIFPGDGSRA GVVH GAIGGA GVTALLALCL CLIFFIV KTH RRKAARTAVG RNDTHPTTGSASPKHQKKSK LHGPTETSSC SGAAPTVEMD EELHYASLNF HGMNPSKDTS TEYSEVRTQ

The amino acid sequence of the extracellular portion of CD33 comprisinga deletion of residues K250 through R254 is provided by SEQ ID NO: 5.The signal peptide is shown in italics and the transmembrane domain isshown in italics with underline.

(SEQ ID NO: 5)MPLLLLLPLL WAGALAMDPN FWLQVQESVT VQEGLCVLVP CTFFHPIPYY DKNSPVHGYWFREGAIISRD SPVATNKLDQ EVQEETQGRF RLLGDPSRNN CSLSIVDARR RDNGSYFFRMERGSTKYSYK SPQLSVHVTD LTHRPKILIP GTLEPGHSKN LTCSVSWACE QGTPPIFSWLSAAPTSLGPR TTHSSVLIIT PRPQDHGTNL TCQVKFAGAG VTTERTIQLN VTYVPQNPTTGIFPGDGSGA GVVH GAIGGA GVTALLALCL CLIFFIV KTH RRKAARTAVG RNDTHPTTGSASPKHQKKSK LHGPTETSSC SGAAPTVEMD EELHYASLNF HGMNPSKDTS TEYSEVRTQ

The amino acid sequence of the extracellular portion of CD33 comprisinga deletion of residues P48 through K52 is provided by SEQ ID NO: 6. Thesignal peptide is shown in italics and the transmembrane domain is shownin italics with underline.

(SEQ ID NO: 6)MPLLLLLPLL WAGALAMDPN FWLQVQESVT VQEGLCVLVP CTFFHPINSP VHGYWFREGAIISRDSPVAT NKLDQEVQEE TQGRFRLLGD PSRNNCSLSI VDARRRDNGS YFFRMERGSTKYSYKSPQLS VHVTDLTHRP KILIPGTLEP GHSKNLTCSV SWACEQGTPP IFSWLSAAPTSLGPRTTHSS VLIITPRPQD HGTNLTCQVK FAGAGVTTER TIQLNVTYVP QNPTTGIFPGDGSGKQETRA GVVH GAIGGA   GVTALLALCL   CLIFFIV KTH RRKAARTAVG RNDTHPTTGSASPKHQKKSK LHGPTETSSC SGAAPTVEMD EELHYASLNF HGMNPSKDTS TEYSEVRTQ

The amino acid sequence of the extracellular portion of CD33 comprisinga deletion of residues Q251 through A255 is provided by SEQ ID NO: 7.The signal peptide is shown in italics and the transmembrane domain isshown in italics with underline.

(SEQ ID NO: 7)MPLLLLLPLL WAGALAMDPN FWLQVQESVT VQEGLCVLVP CTFFHPIPYY DKNSPVHGYWFREGAIISRD SPVATNKLDQ EVQEETQGRF RLLGDPSRNN CSLSIVDARR RDNGSYFFRMERGSTKYSYK SPQLSVHVTD LTHRPKILIP GTLEPGHSKN LTCSVSWACE QGTPPIFSWLSAAPTSLGPR TTHSSVLIIT PRPQDHGTNL TCQVKFAGAG VTTERTIQLN VTYVPQNPTTGIFPGDGSGK GVVH GAIGGA GVTALLALCL CLIFFIV KTH RRKAARTAVG RNDTHPTTGSASPKHQKKSK LHGPTETSSC SGAAPTVEMD EELHYASLNF HGMNPSKDTS TEYSEVRTQ

In some examples, provided herein are variants of CD33, which maycomprise a deletion or mutation of a fragment of the protein that isencoded by any one of the exons of CD33, or a deletion or mutation in anon-essential epitope. The predicted structure of CD33 includes twoimmunoglobulin domains, an IgV domain and an IgC2 domain. In someembodiments, a portion of the immunoglobulin V domain of CD33 is deletedor mutated. In some embodiments, a portion of the immunoglobulin Cdomain of CD33 is deleted or mutated. In some embodiments, exon 2 ofCD33 is deleted or mutated. In some embodiments, the CD33 variant lacksamino acid residues W11 to T139 of SEQ ID NO: 1. In some embodiments,the CD33 variant lacks amino acid residues G13 to T139 of SEQ ID NO: 1.In some embodiments, the deleted or mutated fragment overlaps orencompasses the epitope to which the cytotoxic agent binds. As describedin Example 1, in some embodiments, the epitope comprises amino acids47-51 or 248-252 of the extracellular portion of CD33 (SEQ ID NO: 1). Insome embodiments, the epitope comprises amino acids 248-252 (SEQ ID NO:8), 47-51 (SEQ ID NO: 9), 249-253 (SEQ ID NO: 10), 250-254 (SEQ ID NO:11), 48-52 (SEQ ID NO: 12), or 251-255 (SEQ ID NO: 13) of theextracellular portion of CD33 (SEQ ID NO: 1).

In some embodiments, the genetically engineered hematopoietic stem cellshave genetic edits in a CD33 gene, wherein exon2 of CD33 is mutated ordeleted. In some embodiments, exon 2 of CD33 is mutated or deleted (seethe amino acid sequence of the exon 2-encoded fragment above). In someembodiments, the genetically engineered hematopoietic stem cells havegenetic edits in a CD33 gene resulting in expression of CD33 withdeleted or mutated exon 2 of CD33. In some embodiments, geneticallyengineered hematopoietic stem cells express CD33, in which exon 2 ofCD33 is mutated or deleted. In some examples, the genetically engineeredhematopoietic cells have a genetically engineered CD33 gene (e.g., agenetically engineered endogenous CD33 gene), wherein the engineeringresults in expression of a CD33 variant having the fragment encoded byexon 2 deleted (CD33ex2).

In some embodiments, the CD33 genes edited HSCs expressing CD33 varianthaving the fragment encoded by exon 2 deleted also comprise a partial orcomplete deletion in the adjacent introns (intron 1 and intron 2) inaddition to the deletion of exon 2.

Exemplary amino acid sequence of CD33 mutants, with fragments G13-T139deleted, is provided below (the junction of exon 1-encoded fragment andexon-3 encoded fragment is shown in boldface):

(SEQ ID NO: 56) MPLLLLLPLLWADLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ (SEQ ID NO: 58)MPLLLLLPLLWADLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPVR

While certain cells may express CD33 proteins lacking the fragmentencoded by exon 2, the genetically engineered hematopoietic cells aredifferent from such native cells in at least the aspect that these cellshave undergone genome editing to modify a CD33 gene such as anendogenous CD33 gene. In other words, the parent hematopoietic stemcells for producing the genetically engineered HSCs carry a CD33 genethat produces exon 2-containing transcripts.

Genetically engineered hematopoietic stem cells carrying an edited CD33gene that expresses this CD33 mutant are also within the scope of thepresent disclosure. Such cells may be a homogenous population containingcells expressing the same CD33 mutant (e.g., CD33ex2). Alternatively,the cells may be a heterogeneous population containing cells expressingdifferent CD33 mutants (which may due to heterogeneous editing/repairingevents inside cells) or cells that do not express CD33 (CD33KO). Inspecific examples, the genetically engineered HSCs may be aheterogeneous population containing cells expressing CD33ex2 and cellsthat do not express CD33 (CD33KO).

Genetically engineered hematopoietic stem cells having edited a CD33gene can be prepared by a suitable genome editing method, such as thoseknown in the art or disclosed herein. In some embodiments, thegenetically engineered hematopoietic stem cells described herein can begenerated using the CRISPR approach. See discussions herein. In certainexamples, specific guide RNAs targeting a fragment of the CD33 gene (anexon sequence or an intron sequence) can be used in the CRISPR method.Exemplary gRNAs for editing the CD33 gene (e.g., deletion of exon 2) areprovided in Example 2, Table 4, and Example 3 below.

In some examples, multiple gRNAs can be used for editing the CD33 genevia CRISPR. Different combinations of gRNAs, e.g., selected from thoselisted in Table 4, can be used in the multiplex approach.

A CD33 pseudogene, known as SIGLEC22P (Gene ID 114195), is locatedupstream of the CD33 gene and shares a certain degree of sequencehomology with the CD33 gene. gRNAs that cross-target regions in thepseudogene and regions in the CD33 gene may lead to production ofaberrant gene products. Thus, in some embodiments, the gRNAs used inmethods of editing CD33 via CRISPR preferably have low or nocross-reactivity with regions inside the pseudogene. In some instances,the gRNAs used in methods of editing CD33 via CRISPR preferably have lowor no cross-reactivity with region(s) in Exon 1, intron 1 or Exon 2 ofCD33 that are homologous to the pseudogene. Such gRNAs can be designedby comparing the sequences of the pseudogene and the CD33 gene to choosetargeting sites inside the CD33 gene that have less or no homology toregions of the pseudogene.

In one example, the pair of gRNA 18 (TTCATGGGTACTGCAGGGCA; SEQ ID NO:44)) and gRNA 24 (GTGAGTGGCTGTGGGGAGAG; SEQ ID NO: 50) are used forediting CD33 via CRISPR. Also provided herein are methods of geneticallyediting CD33 in hematopoietic cells (e.g., HSCs) via CRISPR, using oneor more of the gRNAs described herein, for example, the pair ofgRNA18+gRNA24. As described herein, the length of a gRNA sequence may bemodified (increased or decreased), for example, to enhance editingspecificity and/or efficiency. In some embodiments, the length of gRNA24 is 20 base pairs. In some embodiments, reducing the length of gRNA 24may reduce the editing efficiency of gRNA 24 for CD33.

Because of the mechanism of Cas9 cutting and DNA repair, there will be aspectrum of repair events including small insertions on 1-2 nucleotidesand occasionally longer deletions. Representative sequences of repairedCD33exon 2 deletion products (intron 1-intron 2 displayed) are shownbelow (SEQ ID NOs:59-65):

Example Comment Repair Sequence Length 1. Ligation:CCCTGCTGTGGGCAGgtgagtggctgtggggagcagggctgggatgggaccct  0 2. Insertion:CCCTGCTGTGGGCAGgtgaAtggctgCggggagcagggctgggatgggaccc +1 3. Insertion:CCCTGCTGTGGGCAGgtgagtggctgtgggcaggtgagtggctgggatgggaccct  +6/−3 4.Insertion: CCCTGCTGTGGGCAGgtgaAtggctgCggggTactgcagggcagggctgggatgggaccct+8 5. Deletion: CCCTGCTGTGGGCAGgtgaAt--------------   ggctggatgggaccct +1/−14 6. Deletion:CCCTGCTGTGGGCAGgtgaatggctg    -------cagggctgggatgggaccct −7 7.Deletion: CCCTGCTGTGGG---------------------------    ctgggatgggaccct −2        −3nt in exo                                                          n 1Despite the heterogeneity at the genomic DNA level, the RNA transcriptsprovided from the edited CD33 gene all encode CD33 mutants having thefragment encoded by exon 2 deleted.

(iii) Genetically Engineered Hematopoietic Cells Expressing Both CD19and CD33 Mutants

Also provided herein are a genetically engineered hematopoietic cellssuch as HSCs that have both the CD19 and CD33 genes edited. In someembodiments, provide herein is a population of genetically engineeredhematopoietic HSCs in which at least 50% of the cells carry geneticallyedited CD19 and CD33 cells in at least on chromosome.

In some embodiments, the edited CD19 gene is capable of expressing aCD19 mutant having the fragment encoded by exon 2 deleted. Alternativelyor in addition, the edited CD33 gene is capable of expressing a CD33mutant having the fragment encoded by exon 3 deleted.

The genetically engineered hematopoietic cells having both CD19 and CD33genes edited can be prepared by conventional methods. In someembodiments, such cells are prepared by CRISPR using a pair of gRNAs,one targeting CD19 and the other targeting CD33. Examples are providedin Example 3, Table 8 below. In one example, the pair of gRNAs can beintroduced into parent HSCs simultaneously and cells having geneticedits in CD19 and/or CD33 can be harvested for further use.

Masked Lineage-Specific Cell Surface Antigens

While many of the embodiments described herein involve mutations to theendogenous genes encoding lineage-specific cell surface antigens, it isunderstood that other approaches may be used instead of or in additionto mutation. For instance, a lineage-specific cell surface antigen canbe masked, e.g., to prevent or reduce its recognition by animmunotherapeutic agent. In some embodiments, masking is used on alineage-specific cell surface antigen that is difficult to mutate, e.g.,because mutation of the gene is inefficient or is deleterious to cellsexpressing the mutant. In some embodiments, the lineage-specific cellsurface protein is CD45. In some embodiments, masking is performed on acell type described herein, e.g., an HSC or HPC.

In some embodiments, masking is accomplished by expressing a maskingprotein in the cell of interest (e.g., by stably expressing DNA encodingthe masking protein in the cell). In some embodiments, the maskingprotein comprises a binding domain (e.g., an antibody or antigen-bindingfragment, e.g., an scFv) that binds the lineage-specific cell surfaceprotein, e.g., in a way that reduces binding of an immunotherapeuticagent to the lineage-specific cell surface protein, e.g., by competingfor binding at the same epitope. In some embodiments, the binding domainbinds CD45. In some embodiments, the making protein further comprisesone or more sequences that direct its localization to the surface of thecell. In some embodiments, the masking protein comprises a transmembranedomain fused to the binding domain. The masking protein may comprise alinker disposed between the transmembrane domain and the binding domain.The masking protein may be expressed at a level that binds to asufficient amount of the lineage-specific cells surface antigen that animmunotherapeutic agent displays reduced binding to and/or reducedkilling of a cell expressing the masking protein compared to anotherwise similar cell that does not express the masking protein.

In some embodiments, a cell described herein has reduced binding to(and/or reduced killing by) two different immunotherapeutic agents thatrecognize two different lineage-specific cell surface antigens. Forinstance, the cell may have a mutation at a gene encoding a firstlineage-specific cell surface antigen, and may comprise a maskingprotein that masks a second lineage-specific cell surface antigen. Insome embodiments, the cell may comprise a first masking protein thatmasks a first lineage-specific cell surface antigen and a second maskingprotein that masks a second lineage-specific cell surface antigen. Insome embodiments, the first and second lineage-specific cell surfaceantigens are antigens listed in Table 1A.

Cells Altered at One or More Lineage-Specific Cell Surface Antigens

While many of the embodiments described herein involve two or morelineage-specific cell surface antigens, the application also disclosesvarious cells altered with respect to a single lineage-specific cellsurface antigen. For instance, the disclosure describes cells mutated atany one of the lineage-specific cell surface antigens described herein(e.g., mutated at one or both alleles of the lineage-specific cellsurface antigen). The disclosure also describes cells expressing asingle masking protein for a single lineage-specific cell surfaceantigen.

II. Cytotoxic Agents Specific to Lineage-Specific Cell-Surface Antigens

Cytotoxic agents targeting cells (e.g., cancer cells) expressing alineage-specific cell-surface antigen can be co-used with thegenetically engineered hematopoietic cells as described herein. As usedherein, the term “cytotoxic agent” refers to any agent that can directlyor indirectly induce cytotoxicity of a target cell, which expresses thelineage-specific cell-surface antigen (e.g., a target cancer cell). Sucha cytotoxic agent may comprise a protein-binding fragment that binds andtargets an epitope of the lineage-specific cell-surface antigen. In someinstances, the cytotoxic agent may comprise an antibody, which may beconjugated to a drug (e.g., an anti-cancer drug) to form anantibody-drug conjugate (ADC).

The cytotoxic agent for use in the methods described herein may directlycause cell death of a target cell. For example, the cytotoxic agent canbe an immune cell (e.g., a cytotoxic T cell) expressing a chimericreceptor. Upon engagement of the protein binding domain of the chimericreceptor with the corresponding epitope in a lineage-specificcell-surface antigen, a signal (e.g., activation signal) may betransduced to the immune cell resulting in release of cytotoxicmolecules, such as peroforins and granzymes, as well as activation ofeffector functions, leading to death of the target cell. In anotherexample, the cytotoxic agent may be an ADC molecule. Upon binding to atarget cell, the drug moiety in the ADC would exert cytotoxic activity,leading to target cell death.

In other embodiments, the cytotoxic agent may indirectly induce celldeath of the target cell. For example, the cytotoxic agent may be anantibody, which, upon binding to the target cell, would trigger effectoractivities (e.g., ADCC) and/or recruit other factors (e.g.,complements), resulting in target cell death.

Any of the cytotoxic agents described herein target a lineage-specificcell-surface antigen, e.g., comprising a protein-binding fragment thatspecifically binds an epitope in the lineage-specific protein.

For leukemias that become resistance to CAR-T therapy, an emergingstrategy is to simultaneously target alternative or multiple antigens(see e.g., Nature Reviews Immunology (2019), Volume 19, pages 73-74 andCancer Discov. (2018) October; 8(10):1219-1226).

In some embodiments, more than one (e.g., 2, 3, 4, 5 or more) cytotoxicagent is used to target more than one (e.g., 1, 2, 3, 4, 5 or more)epitopes of a lineage-specific cell-surface antigen. In someembodiments, more than one (e.g., 1, 2, 3, 4, 5 or more) cytotoxic agentis used to target an epitope(s) of one or more lineage-specificcell-surface antigen(s) (e.g., additional/alternative antigens). In someembodiments, targeting of more than one lineage-specific cell-surfaceantigen reduces relapse of a hematopoietic malignancy. In oneembodiment, two or more cytotoxic agents are used in the methodsdescribed herein. In some embodiments, the two or more cytotoxic agentsare administered concurrently. In some embodiments, the two or morecytotoxic agents are administered sequentially.

Examples of additional cell-surface proteins that may be targeted areknown in the art (see, e.g., Tasian, Ther. Adv. Hematol. (2018) 9(6):135-148; Hoseini and Cheung, Blood Cancer Journal (2017) 7, e522;doi:10.1038/bcj.2017.2; Taraseviciute et al. Hematology and Oncology(2019) 31(1)). In some embodiments, the methods described herein involvetargeting a lineage-specific cell-surface antigen and one or moreadditional cell-surface proteins. In some embodiments, the methodsdescribed herein involved administering a cytotoxic agent targeting CD33and at least one additional cytotoxic agent that targets an additionalcell-surface protein, such as CD7, CD13, CD15, CD25 (IL-2Ra), CD30, CD32(FcγRIII), CD38, CD44v6, CD45, CD47, CD56, CD90 (Thy1), CD96, CD117(c-KIT), CD123 (IL3Rα), CD135 (FLT3R), CD174 (Lewis-Y), CLL-1 (CLEC12A),folate receptor-b, IL1RAP, MUC1, NKG2D/NKG2DL, TIM-3 (HAVCR2), CD19, andWT1. In some embodiments, the methods described herein involvedadministering a cytotoxic agent targeting CD19 and at least oneadditional cytotoxic agent that targets an additional cell-surfaceprotein, such as CD7, CD13, CD15, CD25 (IL-2Rα), CD30, CD32 (FcγRIII),CD38, CD44v6, CD45, CD47, CD56, CD90 (Thy1), CD96, CD117 (c-KIT), CD123(IL3Rα), CD135 (FLT3R), CD174 (Lewis-Y), CLL-1 (CLEC12A), folatereceptor-b, IL1RAP, MUC1, NKG2D/NKG2DL, TIM-3 (HAVCR2), CD19, and WT1.

In some examples, a cytotoxic agent is used to target CD33 and a secondcytotoxic agent is used to target CD19. In some examples, a cytotoxicagent is used to target CD33 and a second cytotoxic agent is used totarget an additional cell-surface protein. In some examples, a cytotoxicagent is used to target CD19 and a second cytotoxic agent is used totarget an additional cell-surface protein. In some examples, a cytotoxicagent is used to target CD33 and a second cytotoxic agent is used totarget CD13. In some examples, a cytotoxic agent is used to target CD33and a second cytotoxic agent is used to target CD13. In some examples, acytotoxic agent is used to target CD33 and a second cytotoxic agent isused to target CD123. In some examples, a cytotoxic agent is used totarget CD19 and a second cytotoxic agent is used to target CD13. In someexamples, a cytotoxic agent is used to target CD19 and a secondcytotoxic agent is used to target CD123. In some examples, a cytotoxicagent is used to target CD13 and a second cytotoxic agent is used totarget CD123.

In some examples, a cytotoxic agent is used to target CD33, a secondcytotoxic agent is used to target CD19, and a third cytotoxic agent isused to target CD13. In some examples, a cytotoxic agent is used totarget CD33, a second cytotoxic agent is used to target CD19, and athird cytotoxic agent is used to target CD123. In some examples, acytotoxic agent is used to target CD33, a second cytotoxic agent is usedto target CD13, and a third cytotoxic agent is used to target CD123. Insome examples, a cytotoxic agent is used to target CD19, a secondcytotoxic agent is used to target CD19, and a third cytotoxic agent isused to target CD13. In some examples, a cytotoxic agent is used totarget CD33, a second cytotoxic agent is used to target CD19, a thirdcytotoxic agent is used to target CD13, and fourth cytotoxic agent usedto target CD123.

(i) Therapeutic Antibodies

Any antibody or an antigen-binding fragment thereof can be used as acytotoxic agent or for constructing a cytotoxic agent that targets anepitope of a lineage-specific cell-surface antigen, as described herein.Such an antibody or antigen-binding fragment can be prepared by aconventional method, for example, the hybridoma technology orrecombinant technology.

As used herein, the term “antibody” refers to a glycoprotein comprisingat least two heavy (H) chains and two light (L) chains inter-connectedby disulfide bonds, i.e., covalent heterotetramers comprised of twoidentical Ig H chains and two identical L chains that are encoded bydifferent genes. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as HCVR or VH) and a heavy chain constantregion. The heavy chain constant region is comprised of three domains,CH1, CH2 and CH3. Each light chain is comprised of a light chainvariable region (abbreviated herein as LCVR or VL) and a light chainconstant region. The light chain constant region is comprised of onedomain, CL. The VH and VL regions can be further subdivided into regionsof hypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system. Formation of amature functional antibody molecule can be accomplished when twoproteins are expressed in stoichiometric quantities and self-assemblewith the proper configuration.

In some embodiments, the antigen-binding fragment is a single-chainantibody fragment (scFv) that specifically binds the epitope of thelineage-specific cell-surface antigen. In other embodiments, theantigen-binding fragment is a full-length antibody that specificallybinds the epitope of the lineage-specific cell-surface antigen.

As described herein and as will be evident to a skilled artisan, theCDRs of an antibody specifically bind to the epitope of a targetprotein/antigen (the lineage-specific cell-surface protein/antigen).

In some embodiments, the antibodies are full-length antibodies, meaningthe antibodies comprise a fragment crystallizable (Fc) portion and afragment antigen-binding (Fab) portion. In some embodiments, theantibodies are of the isotype IgG, IgA, IgM, IgA, or IgD. In someembodiments, a population of antibodies comprises one isotype ofantibody. In some embodiments, the antibodies are IgG antibodies. Insome embodiments, the antibodies are IgM antibodies. In someembodiments, a population of antibodies comprises more than one isotypeof antibody. In some embodiments, a population of antibodies iscomprised of a majority of one isotype of antibodies but also containsone or more other isotypes of antibodies. In some embodiments, theantibodies are selected from the group consisting of IgG1, IgG2, IgG3,IgG4, IgM, IgA1, IgA2, IgAsec, IgD, IgE.

The antibodies described herein may specifically bind to a targetprotein. As used herein, “specific binding” refers to antibody bindingto a predetermined protein, such as a cancer antigen. “Specific binding”involves more frequent, more rapid, greater duration of interaction,and/or greater affinity to a target protein relative to alternativeproteins. In some embodiments, a population of antibodies specificallybinds to a particular epitope of a target protein, meaning theantibodies bind to the particular protein with more frequently, morerapidly, for greater duration of interaction, and/or with greateraffinity to the epitope relative to alternative epitopes of the sametarget protein or to epitopes of another protein. In some embodiments,the antibodies that specifically bind to a particular epitope of atarget protein may not bind to other epitopes of the same protein.

Antibodies may be selected based on the binding affinity of the antibodyto the target protein or epitope. Alternatively or in additional, theantibodies may be mutated to introduce one or more mutations to modify(e.g., enhance or reduce) the binding affinity of the antibody to thetarget protein or epitope.

The present antibodies or antigen-binding portions can specifically bindwith a dissociation constant (K_(D)) of less than about 10⁻⁷ M, lessthan about 10⁻⁸ M, less than about 10⁻⁹ M, less than about 10⁻¹⁰ M, lessthan about 10⁻¹¹ M, or less than about 10⁻¹² M. Affinities of theantibodies according to the present disclosure can be readily determinedusing conventional techniques (see, e.g., Scatchard et al., Ann. N.Y.Acad. Sci. (1949) 51:660; and U.S. Pat. Nos. 5,283,173, 5,468,614, orthe equivalent).

The binding affinity or binding specificity for an epitope or proteincan be determined by a variety of methods including equilibriumdialysis, equilibrium binding, gel filtration, ELISA, surface plasmonresonance, or spectroscopy.

For example, antibodies (of antigen-binding fragments thereof) specificto an epitope of a lineage-specific protein of interest can be made bythe conventional hybridoma technology. The lineage-specific protein,which may be coupled to a carrier protein such as KLH, can be used toimmunize a host animal for generating antibodies binding to thatcomplex. The route and schedule of immunization of the host animal aregenerally in keeping with established and conventional techniques forantibody stimulation and production, as further described herein.General techniques for production of mouse, humanized, and humanantibodies are known in the art and are described herein. It iscontemplated that any mammalian subject including humans or antibodyproducing cells therefrom can be manipulated to serve as the basis forproduction of mammalian, including human hybridoma cell lines.Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, and/orintradermally with an amount of immunogen, including as describedherein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D.W., et al., In Vitro, 18:377-381 (1982). Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute®, Cell Distribution Center, San Diego, Calif., USA, may beused in the hybridization. Generally, the technique involves fusingmyeloma cells and lymphoid cells using a fusogen such as polyethyleneglycol, or by electrical means well known to those skilled in the art.After the fusion, the cells are separated from the fusion medium andgrown in a selective growth medium, such ashypoxanthine-aminopterin-thymidine (HAT) medium, to eliminateunhybridized parent cells. Any of the media described herein,supplemented with or without serum, can be used for culturing hybridomasthat secrete monoclonal antibodies. As another alternative to the cellfusion technique, EBV immortalized B cells may be used to produce theTCR-like monoclonal antibodies described herein. The hybridomas areexpanded and subcloned, if desired, and supernatants are assayed foranti-immunogen activity by conventional immunoassay procedures (e.g.,radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies capable of binding to a lineage-specific protein.Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen. Immunization of a host animal with a target protein or afragment containing the target amino acid sequence conjugated to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example maleimidobenzoyl sulfosuccinimide ester (conjugation throughcysteine residues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl, or R1N═C═NR, where R and R1are different alkyl groups, can yield a population of antibodies (e.g.,monoclonal antibodies).

If desired, an antibody of interest (e.g., produced by a hybridoma) maybe sequenced and the polynucleotide sequence may then be cloned into avector for expression or propagation. The sequence encoding the antibodyof interest may be maintained in vector in a host cell and the host cellcan then be expanded and frozen for future use. In an alternative, thepolynucleotide sequence may be used for genetic manipulation to“humanize” the antibody or to improve the affinity (affinitymaturation), or other characteristics of the antibody. For example, theconstant region may be engineered to more resemble human constantregions to avoid immune response if the antibody is used in clinicaltrials and treatments in humans. It may be desirable to geneticallymanipulate the antibody sequence to obtain greater affinity to thelineage-specific protein. In some examples, the antibody sequence ismanipulated to increase binding affinity of the antibody to thelineage-specific protein such that lower levels of the lineage-specificprotein are detected by the antibody. In some embodiments, antibodiesthat have increased binding to the lineage-specific protein may be usedto reduce or prevent relapse of a hematopoietic malignancy. It will beapparent to one of skill in the art that one or more polynucleotidechanges can be made to the antibody and still maintain its bindingspecificity to the target protein.

In other embodiments, fully human antibodies can be obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse® fromAmgen®, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex®, Inc. (Princeton, N.J.). In another alternative, antibodies maybe made recombinantly by phage display or yeast technology. See, forexample, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150;and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively,the phage display technology (McCafferty et al., (1990) Nature348:552-553) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors.

Antigen-binding fragments of an intact antibody (full-length antibody)can be prepared via routine methods. For example, F(ab′)₂ fragments canbe produced by pepsin digestion of an antibody molecule, and Fabfragments that can be generated by reducing the disulfide bridges ofF(ab′)2 fragments.

Genetically engineered antibodies, such as humanized antibodies,chimeric antibodies, single-chain antibodies, and bi-specificantibodies, can be produced via, e.g., conventional recombinanttechnology. In one example, DNA encoding a monoclonal antibodiesspecific to a target protein can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the monoclonal antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoone or more expression vectors, which are then transfected into hostcells such as E. coli cells, simian COS cells, Chinese hamster ovary(CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. See, e.g., PCT Publication No. WO87/04462. The DNA can then be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences, Morrison et al., (1984) Proc.Nat. Acad. Sci. 81:6851, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, genetically engineeredantibodies, such as “chimeric” or “hybrid” antibodies; can be preparedthat have the binding specificity of a target protein.

Techniques developed for the production of “chimeric antibodies” arewell known in the art. See, e.g., Morrison et al. (1984) Proc. Natl.Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; andTakeda et al. (1984) Nature 314:452.

Methods for constructing humanized antibodies are also well known in theart. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033(1989). In one example, variable regions of VH and VL of a parentnon-human antibody are subjected to three-dimensional molecular modelinganalysis following methods known in the art. Next, framework amino acidresidues predicted to be important for the formation of the correct CDRstructures are identified using the same molecular modeling analysis. Inparallel, human VH and VL chains having amino acid sequences that arehomologous to those of the parent non-human antibody are identified fromany antibody gene database using the parent VH and VL sequences assearch queries. Human VH and VL acceptor genes are then selected.

The CDR regions within the selected human acceptor genes can be replacedwith the CDR regions from the parent non-human antibody or functionalvariants thereof. When necessary, residues within the framework regionsof the parent chain that are predicted to be important in interactingwith the CDR regions (see above description) can be used to substitutefor the corresponding residues in the human acceptor genes.

A single-chain antibody can be prepared via recombinant technology bylinking a nucleotide sequence coding for a heavy chain variable regionand a nucleotide sequence coding for a light chain variable region.Preferably, a flexible linker is incorporated between the two variableregions. Alternatively, techniques described for the production ofsingle chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can beadapted to produce a phage or yeast scFv library and scFv clonesspecific to a lineage-specific protein can be identified from thelibrary following routine procedures. Positive clones can be subjectedto further screening to identify those that bind lineage-specificprotein.

In some instances, the cytotoxic agent for use in the methods describedherein comprises an antigen-binding fragment that targets thelineage-specific protein CD33. In other examples, the cytotoxic agentfor use in the methods described herein comprises an antigen-bindingfragment that targets the lineage-specific protein CD19. In otherexample, two or more cytotoxic agents are used in the methods describedherein. In some embodiments, the two or more cytotoxic agents areadministered concurrently. In some embodiments, the two or morecytotoxic agents are administered sequentially. In one non-limitingexample, antibodies and antigen-binding fragments targeting CD33 andCD19 in combination are used in the methods described herein. In onenon-limiting example, antibodies and antigen-binding fragments targetingCD33 are used in combination with a cytotoxic agent (e.g., antibodies,immune cells expressing chimeric antigen receptors, antibody-drugconjugates) that targets a second lineage-specific cell-surface antigenor an additional cell-surface protein. In one non-limiting example,antibodies and antigen-binding fragments targeting CD19 are used incombination with a cytotoxic agent (e.g., antibodies, immune cellsexpressing chimeric antigen receptors, antibody-drug conjugates) thattargets a second lineage-specific cell-surface antigen or an additionalcell-surface protein.

In some embodiments, bispecific or multi-specific antibodies may be usedto target more than one epitope (e.g., more than one epitope of alineage-specific cell-surface antigen, epitopes of more than onelineage-specific cell-surface antigen, an epitope of lineage-specificcell-surface antigen and an epitope of an additional cell-surfaceantigen). See, e.g., Hoseini et al. Blood Cancer Journal (2017) 7, e552.Non-limiting examples of bispecific antibodies include tandem doublescFv (e.g., single-chain bispecific tandem fragment variable (scBsTaFv),bispecific T-cell engager (BiTE), bispecific single-chain Fv (bsscFv),bispecific killer-cell engager (BiKE), dual-affinity re-targeting(DART), diabody, tandem diabodies (TandAb), single-chain Fv triplebody(sctb), bispecific scFv immunofusion (Blf), Fabsc, dual-variable-domainimmunoglobulin (DVD-Ig), CrossMab (CH1-CL), modular bispecific antibody(IgG-scFv). See, e.g., Marin-Acevedo et al. J. Hematol. Oncol. (2018)11:8; Slaney et al. Cancer Discovery (2018) 8(8): 924-934, and Elgundi etal. Advanced Drug Discovery Reviews (2017) 122: 2-19.

In some embodiments, the antibody is a bispecific T-cell engager (BiTE)comprising two linked scFv molecules. In some embodiments, at least ofthe linked scFv of the BiTE binds an epitope of a lineage-specificcell-surface protein (e.g., CD33 or CD19). In one example, the BiTE isblinatumomab. See, e.g. Slaney et al. Cancer Discovery (2018) 8(8):924-934.

For example, an antibody that targets both CD33 and CD19 may be used inthe methods described herein. Antibodies and antigen-binding fragmentstargeting CD33 or CD19 or a combination thereof can be prepared byroutine practice. Non-limited examples of antigen-binding fragments thattarget CD19 can be found in Porter D L et al. NEJM (2011) 365:725-33 andKalos M et al. Sci Transl Med. (2011) 3:95ra73. See also descriptionsherein. Such CD19-targeting antigen-binding fragments can be used formaking the CAR constructs described herein.

In some embodiments, a bispecific antibody may be used in which onemolecule targets an epitope of a lineage-specific cell-surface proteinon a target cell and the other molecule targets a surface antigen on aneffector cell (e.g., T cell, NK cell) such that the target cell isbrought into proximity with the effector cell. See, e.g., Hoseini et al.Blood Cancer Journal (2017) 7, e552.

In some embodiments, two or more (e.g., 2, 3, 4, 5 or more) epitopes ofa lineage-specific cell-surface protein have been modified, enabling twoor more (e.g., 2, 3, 4, 5 or more) different cytotoxic agents (e.g., twoantibodies) to be targeted to the two or more epitopes. In someembodiments, the antibodies could work synergistically to enhanceefficacy. In some embodiments, epitopes of two or more (e.g., 2, 3, 4, 5or more) lineage-specific cell surface protein have been modified,enabling two or more (e.g., 2, 3, 4, 5 or more) different cytotoxicagents (e.g., two antibodies) to be targeted to epitopes of the two ormore lineage-specific cell-surface proteins. In some embodiments, one ormore (e.g., 1, 2, 3, 4, 5 or more) epitopes of a lineage-specificcell-surface protein have been modified and one or more (e.g., 1, 2, 3,4, 5 or more) epitopes of an additional cell-surface protein have beenmodified, enabling two or more (e.g., 2, 3, 4, 5 or more) differentcytotoxic agents (e.g., two antibodies) to be targeted to epitopes ofthe lineage-specific cell-surface protein and epitopes of additionalcell-surface protein. In some embodiments, targeting of two or morelineage-specific cell-surface protein may reduce relapse of ahematopoietic malignancy.

In some embodiments, the methods described herein involve administeringa cytotoxic agent that targets an epitope of a lineage-specificcell-surface antigen that is mutated in the population of geneticallyengineered hematopoietic cells. In some embodiments, the methodsdescribed herein involve administering a cytotoxic agent that targets anepitope of a lineage-specific cell-surface antigen that is mutated inthe population of genetically engineered hematopoietic cells and one ormore additional cytotoxic agents that target one or more additionalcell-surface proteins. In some embodiments, the antibodies worksynergistically to enhance efficacy by targeting more than onecell-surface protein.

In some embodiments, the methods described herein involve administeringto the subject a population of genetically engineered cells lacking anon-essential epitope in a lineage-specific cell-surface antigen and oneor more immunotherapeutic agents (e.g., antibodies) that target cellsexpressing the lineage-specific cell-surface antigen. In someembodiments, the methods described herein involve administering to thesubject a population of genetically engineered cells lacking anon-essential epitope in a type 1 lineage-specific cell-surface antigenand one or more immunotherapeutic agents (e.g., antibodies) that targetcells expressing the lineage-specific cell-surface antigen. In someembodiments, the methods described herein involve administering to thesubject a population of genetically engineered cells lacking anon-essential epitope in a type 2 lineage-specific cell-surface antigenand one or more immunotherapeutic agents (e.g., antibodies) that targetcells expressing the lineage-specific cell-surface antigen. In any ofthe embodiments described herein, one or more additionalimmunotherapeutic agents may be further administered to the subject(e.g., targeting one or more additional epitopes and/or antigens), forexample if the hematopoietic malignancy relapses.

In some examples, the methods described herein involve administering tothe subject a population of genetically engineered cells lacking anon-essential epitope of CD33 and one or more antibodies that targetcells expressing CD33. In some examples, the methods described hereininvolve administering to the subject a population of geneticallyengineered cells lacking an epitope in exon 2 or exon 3 of CD33 and oneor more antibodies that target cells expressing CD33. In some examples,the methods described herein involve administering to the subject apopulation of genetically engineered cells expressing a mutated CD33comprising the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 58 andone or more antibodies that target cells expressing CD33.

In some examples, the methods described herein involve administering tothe subject a population of genetically engineered cells lacking anon-essential epitope of CD19 and one or more antibodies that targetcells expressing CD19. In some examples, the methods described hereininvolve administering to the subject a population of geneticallyengineered cells lacking an epitope in exon 2 or exon 4 of CD19 and oneor more antibodies that target cells expressing CD19. In some examples,the methods described herein involve administering to the subject apopulation of genetically engineered cells expressing a mutated CD19comprising the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 73 andone or more antibodies that target cells expressing CD19.

(ii) Immune Cells Expressing Chimeric Antigen Receptors

In some embodiments, the cytotoxic agent that targets an epitope of alineage-specific cell-surface antigen as described herein is an immunecell that expresses a chimeric receptor, which comprises anantigen-binding fragment (e.g., a single-chain antibody) capable ofbinding to the epitope of the lineage-specific protein (e.g., CD33 orCD19). Recognition of a target cell (e.g., a cancer cell) having theepitope of the lineage-specific protein on its cell surface by theantigen-binding fragment of the chimeric receptor transduces anactivation signal to the signaling domain(s) (e.g., co-stimulatorysignaling domain and/or the cytoplasmic signaling domain) of thechimeric receptor, which may activate an effector function in the immunecell expressing the chimeric receptor. In some embodiments, the immunecell expresses more than one chimeric receptor (e.g., 2, 3, 4, 5 ormore), referred to as a bispecific or multi-specific immune cell. Insome embodiments, the immune cell expresses more than one chimericreceptor, at least one of which targets an epitope of a lineage-specificcell-surface antigen. In some embodiments, the immune cell expressesmore than one chimeric receptor, each of which targets an epitope of alineage-specific cell-surface antigen. In some embodiments, the immunecell expresses more than one chimeric receptor, at least one of whichtargets an epitope of a lineage-specific cell-surface antigen and atleast one of which targets an epitope of an additional cell-surfaceantigen. In some embodiments, targeting of more than onelineage-specific cell-surface protein or a lineage-specific cell-surfaceprotein and one or more additional cell-surface protein may reducerelapse of a hematopoietic malignancy. In some embodiments, the immunecell expresses a chimeric receptor that targets more than one epitopes(e.g., more than one epitopes of one antigen or epitopes of more thanone antigen), referred to as a bispecific chimeric receptor.

In some embodiments, epitopes of two or more lineage-specificcell-surface proteins are targeted by cytotoxic agents. In someembodiments, two or more chimeric receptors are expressed in the sameimmune cell, e.g., bispecific chimeric receptors. Such cells can be usedin any of the methods described herein. In some embodiments, cellsexpressing a chimeric receptor are “pooled”, i.e., two or more groups ofcells express two or more different chimeric receptors. In someembodiments, two or more cells expressing different chimeric antigenreceptors are administered concurrently. In some embodiments, two ormore cells expressing different chimeric antigen receptors areadministered sequentially. In some embodiments, epitopes of CD33 andCD19 are targeted by cytotoxic agents. In some embodiments, the chimericreceptors targeting CD33 and CD19 are expressed in the same immune cell(i.e., a bispecific immune cell). Such cells can be used in any of themethods described herein. In some embodiments, cells expressing chimericreceptors targeting CD33 and CD19 “pooled”, i.e., two or more groups ofcells express two or more different chimeric receptors. In someembodiments, two or more groups of cells expressing chimeric receptorstargeting CD33 and CD19 are administered concurrently. In someembodiments, two or more groups of cells expressing chimeric receptorstargeting CD33 and CD19 are administered sequentially.

As used herein, a chimeric receptor refers to a non-naturally occurringmolecule that can be expressed on the surface of a host cell andcomprises binding domain that provides specificity of the chimericreceptor (e.g., an antigen-binding fragment that binds to an epitope ofa cell-surface lineage-specific protein). In general, chimeric receptorscomprise at least two domains that are derived from different molecules.In addition to the epitope-binding fragment described herein, thechimeric receptor may further comprise one or more of the following: ahinge domain, a transmembrane domain, a co-stimulatory domain, acytoplasmic signaling domain, and combinations thereof. In someembodiments, the chimeric receptor comprises from N terminus to Cterminus, an antigen-binding fragment that binds to a cell-surfacelineage-specific protein, a hinge domain, a transmembrane domain, and acytoplasmic signaling domain. In some embodiments, the chimeric receptorfurther comprises at least one co-stimulatory domain. See, e.g.,Marin-Acevedo et al. J. Hematol. Oncol. (2018)11: 8.

Alternatively or in addition, the chimeric receptor may be a switchablechimeric receptor. See, e.g., Rodger et al. PNAS (2016) 113: 459-468;Cao et al. Angew. Chem. Int. Ed. (2016) 55: 7520-7524. In general, aswitchable chimeric receptor comprises a binding domain that binds asoluble antigen-binding fragment, which has antigen binding specificityand may be administered concomitantly with the immune cells.

In some embodiments, the chimeric receptor may be a masked chimericreceptor, which is maintained in an “off” state until the immune cellexpressing the chimeric receptor is localized to a desired location inthe subject. For example, the binding domain of the chimeric receptor(e.g., antigen-binding fragment) may be blocked by an inhibitory peptidethat is cleaved by a protease present at a desired location in thesubject.

In some embodiments, it may be advantageous to modulate the bindingaffinity of the binding domain (e.g., antigen-binding fragment). Forexample, in some instances, relapse of hematopoietic malignanciesresults due to the reduced expression of the targeted antigen on thesurface of target cells (e.g., antigen escape) and the lower levels ofantigen any be inefficient or less efficient in stimulating cytotoxicityof the target cells. See, e.g., Majzner et al. Cancer Discovery (2018)8(10). In some embodiments, the binding affinity of the binding domain(e.g., antigen-binding fragment) may be enhanced, for example bymutating one or more amino acid residues of the binding domain. Bindingdomains having enhanced binding affinity to an antigen may result inimmune cells that response to lower levels of antigen (lower antigendensity) and reduce or prevent relapse.

In some embodiments, the chimeric receptors described herein compriseone or more hinge domain(s). In some embodiments, the hinge domain maybe located between the antigen-binding fragment and a transmembranedomain. A hinge domain is an amino acid segment that is generally foundbetween two domains of a protein and may allow for flexibility of theprotein and movement of one or both of the domains relative to oneanother. Any amino acid sequence that provides such flexibility andmovement of the antigen-binding fragment relative to another domain ofthe chimeric receptor can be used.

The hinge domain may contain about 10-200 amino acids, e.g., 15-150amino acids, 20-100 amino acids, or 30-60 amino acids. In someembodiments, the hinge domain may be of about 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, or 200 amino acids in length.

In some embodiments, the hinge domain is a hinge domain of a naturallyoccurring protein. Hinge domains of any protein known in the art tocomprise a hinge domain are compatible for use in the chimeric receptorsdescribed herein. In some embodiments, the hinge domain is at least aportion of a hinge domain of a naturally occurring protein and confersflexibility to the chimeric receptor. In some embodiments, the hingedomain is of CD8α or CD28. In some embodiments, the hinge domain is aportion of the hinge domain of CD8α, e.g., a fragment containing atleast 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of thehinge domain of CD8α or CD28.

Hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgDantibody, are also compatible for use in the chimeric receptorsdescribed herein. In some embodiments, the hinge domain is the hingedomain that joins the constant domains CH1 and CH2 of an antibody. Insome embodiments, the hinge domain is of an antibody and comprises thehinge domain of the antibody and one or more constant regions of theantibody. In some embodiments, the hinge domain comprises the hingedomain of an antibody and the CH3 constant region of the antibody. Insome embodiments, the hinge domain comprises the hinge domain of anantibody and the CH2 and CH3 constant regions of the antibody. In someembodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. Insome embodiments, the antibody is an IgG antibody. In some embodiments,the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In someembodiments, the hinge region comprises the hinge region and the CH2 andCH3 constant regions of an IgG1 antibody. In some embodiments, the hingeregion comprises the hinge region and the CH3 constant region of an IgG1antibody.

Also within the scope of the present disclosure are chimeric receptorscomprising a hinge domain that is a non-naturally occurring peptide. Insome embodiments, the hinge domain between the C-terminus of theextracellular ligand-binding domain of an Fc receptor and the N-terminusof the transmembrane domain is a peptide linker, such as a (Gly_(x)Ser)nlinker (SEQ ID NOs: 124-133), wherein x and n, independently can be aninteger between 3 and 12, including 3 (SEQ ID NO: 124), 4 (SEQ ID NO:125), 5 (SEQ ID NO: 126), 6 (SEQ ID NO: 127), 7 (SEQ ID NO: 128), 8 (SEQID NO: 129), 9 (SEQ ID NO: 130), 10 (SEQ ID NO: 131), 11 (SEQ ID NO:132), 12 (SEQ ID NO: 133), or more.

Additional peptide linkers that may be used in a hinge domain of thechimeric receptors described herein are known in the art. See, e.g.,Wriggers et al. Current Trends in Peptide Science (2005) 80(6): 736-746and PCT Publication WO 2012/088461.

In some embodiments, the chimeric receptors described herein maycomprise one or more transmembrane domain(s). The transmembrane domainfor use in the chimeric receptors can be in any form known in the art.As used herein, a “transmembrane domain” refers to any protein structurethat is thermodynamically stable in a cell membrane, preferably aeukaryotic cell membrane. Transmembrane domains compatible for use inthe chimeric receptors used herein may be obtained from a naturallyoccurring protein. Alternatively, the transmembrane domain may be asynthetic, non-naturally occurring protein segment, e.g., a hydrophobicprotein segment that is thermodynamically stable in a cell membrane.

Transmembrane domains are classified based on the transmembrane domaintopology, including the number of passes that the transmembrane domainmakes across the membrane and the orientation of the protein. Forexample, single-pass membrane proteins cross the cell membrane once, andmulti-pass membrane proteins cross the cell membrane at least twice(e.g., 2, 3, 4, 5, 6, 7 or more times). In some embodiments, thetransmembrane domain is a single-pass transmembrane domain. In someembodiments, the transmembrane domain is a single-pass transmembranedomain that orients the N terminus of the chimeric receptor to theextracellular side of the cell and the C terminus of the chimericreceptor to the intracellular side of the cell. In some embodiments, thetransmembrane domain is obtained from a single pass transmembraneprotein. In some embodiments, the transmembrane domain is of CD8α. Insome embodiments, the transmembrane domain is of CD28. In someembodiments, the transmembrane domain is of ICOS.

In some embodiments, the chimeric receptors described herein compriseone or more costimulatory signaling domains. The term “co-stimulatorysignaling domain,” as used herein, refers to at least a portion of aprotein that mediates signal transduction within a cell to induce animmune response, such as an effector function. The co-stimulatorysignaling domain of the chimeric receptor described herein can be acytoplasmic signaling domain from a co-stimulatory protein, whichtransduces a signal and modulates responses mediated by immune cells,such as T cells, NK cells, macrophages, neutrophils, or eosinophils.

In some embodiments, the chimeric receptor comprises more than one (atleast 2, 3, 4, or more) co-stimulatory signaling domains. In someembodiments, the chimeric receptor comprises more than oneco-stimulatory signaling domains obtained from different costimulatoryproteins. In some embodiments, the chimeric receptor does not comprise aco-stimulatory signaling domain.

In general, many immune cells require co-stimulation, in addition tostimulation of an antigen-specific signal, to promote cellproliferation, differentiation and survival, and to activate effectorfunctions of the cell. Activation of a co-stimulatory signaling domainin a host cell (e.g., an immune cell) may induce the cell to increase ordecrease the production and secretion of cytokines, phagocyticproperties, proliferation, differentiation, survival, and/orcytotoxicity. The co-stimulatory signaling domain of any co-stimulatoryprotein may be compatible for use in the chimeric receptors describedherein. The type(s) of co-stimulatory signaling domain is selected basedon factors such as the type of the immune cells in which the chimericreceptors would be expressed (e.g., primary T cells, T cell lines, NKcell lines) and the desired immune effector function (e.g.,cytotoxicity). Examples of co-stimulatory signaling domains for use inthe chimeric receptors can be the cytoplasmic signaling domain ofco-stimulatory proteins, including, without limitation, CD27, CD28,4-1BB, OX40, CD30, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3. In someembodiments, the co-stimulatory domain is derived from 4-1BB, CD28, orICOS. In some embodiments, the costimulatory domain is derived from CD28and chimeric receptor comprises a second co-stimulatory domain from4-1BB or ICOS.

In some embodiments, the costimulatory domain is a fusion domaincomprising more than one costimulatory domain or portions of more thanone costimulatory domains. In some embodiments, the costimulatory domainis a fusion of costimulatory domains from CD28 and ICOS.

In some embodiments, the chimeric receptors described herein compriseone or more cytoplasmic signaling domain(s). Any cytoplasmic signalingdomain can be used in the chimeric receptors described herein. Ingeneral, a cytoplasmic signaling domain relays a signal, such asinteraction of an extracellular ligand-binding domain with its ligand,to stimulate a cellular response, such as inducing an effector functionof the cell (e.g., cytotoxicity).

As will be evident to one of ordinary skill in the art, a factorinvolved in T cell activation is the phosphorylation of immunoreceptortyrosine-based activation motif (ITAM) of a cytoplasmic signalingdomain. Any ITAM-containing domain known in the art may be used toconstruct the chimeric receptors described herein. In general, an ITAMmotif may comprise two repeats of the amino acid sequence YxxL/Iseparated by 6-8 amino acids, wherein each x is independently any aminoacid, producing the conserved motif YxxL/Ix(6-8)YxxL/I. In someembodiments, the cytoplasmic signaling domain is from CD3ζ.

In some embodiments, the chimeric receptor described herein targets atype 2 protein. In some embodiments, the chimeric receptor targets CD33.In some embodiments, the chimeric receptor described herein targets atype 1 protein. In some embodiments, the chimeric receptor targets CD19.In some embodiments, the chimeric receptor targets a type 0 protein.Such a chimeric receptor may comprise an antigen-binding fragment (e.g.,an scFv) comprising a heavy chain variable region and a light chainvariable region that bind to CD19. Alternatively, the chimeric receptormay comprise an antigen-binding fragment (e.g., scFv) comprising a heavychain variable region and a light chain variable region that bind toCD33.

In some embodiments, the immune cells described herein express achimeric receptor (e.g., bispecific chimeric receptor) that targets atype 2 protein and a chimeric receptor that targets a type 1 protein. Insome embodiments, the immune cells described herein express a chimericreceptor (e.g., bispecific chimeric receptor) that targets a type 2protein and at least one additional cell-surface protein, such as any ofthose described herein. In some embodiments, the immune cells describedherein express a chimeric receptor that targets CD33 and at least oneadditional cell-surface protein, such as any of those described herein.In some embodiments, the immune cells described herein express achimeric receptor that targets a type 1 protein and at least oneadditional cell-surface protein, such as any of those described herein.In some embodiments, the immune cells described herein express achimeric receptor that targets CD19 and at least one additionalcell-surface protein, such as any of those described herein. In someembodiments, the chimeric receptor described herein targets CD33 andCD19.

In some embodiments, the immune cells described herein express achimeric receptor that targets a type 2 protein and at least oneadditional chimeric receptor that targets an additional cell-surfaceprotein, such as any of those described herein. In some embodiments, theimmune cells described herein express a chimeric receptor that targetsCD33 and at least one additional chimeric receptor that targets anothercell-surface protein, such as any of those described herein. In someembodiments, the immune cells described herein express a chimericreceptor that targets a type 1 protein and at least one additionalchimeric receptor that targets another cell-surface protein, such as anyof those described herein. In some embodiments, the immune cellsdescribed herein express a chimeric receptor that targets CD19 and atleast one additional chimeric receptor that targets another cell-surfaceprotein, such as any of those described herein. In some embodiments, theimmune cells express a chimeric receptor that targets CD33 and achimeric receptor that targets CD19.

A chimeric receptor construct targeting CD33 and/or CD19 may furthercomprise at least a hinge domain (e.g., from CD28, CD8α, or anantibody), a transmembrane domain (e.g., from CD8α, CD28 or ICOS), oneor more co-stimulatory domains (from one or more of CD28, ICOS, or4-1BB) and a cytoplasmic signaling domain (e.g., from CD3ζ), or acombination thereof. In some examples, the methods described hereininvolve administering to a subject a population of geneticallyengineered hematopoietic cells and an immune cell expressing a chimericreceptor that targets CD33 and/or CD19, which may further comprise atleast a hinge domain (e.g., from CD28, CD8α, or an antibody), atransmembrane domain (e.g., from CD8α, CD28 or ICOS), one or moreco-stimulatory domains (from one or more of CD28, ICOS, or 4-1BB) and acytoplasmic signaling domain (e.g., from CD3ζ), or a combination thereof

Any of the chimeric receptors described herein can be prepared byroutine methods, such as recombinant technology. Methods for preparingthe chimeric receptors herein involve generation of a nucleic acid thatencodes a polypeptide comprising each of the domains of the chimericreceptors, including the antigen-binding fragment and optionally, thehinge domain, the transmembrane domain, at least one co-stimulatorysignaling domain, and the cytoplasmic signaling domain. In someembodiments, nucleic acids encoding the components of a chimericreceptor are joined together using recombinant technology.

Sequences of each of the components of the chimeric receptors may beobtained via routine technology, e.g., PCR amplification from any one ofa variety of sources known in the art. In some embodiments, sequences ofone or more of the components of the chimeric receptors are obtainedfrom a human cell. Alternatively, the sequences of one or morecomponents of the chimeric receptors can be synthesized. Sequences ofeach of the components (e.g., domains) can be joined directly orindirectly (e.g., using a nucleic acid sequence encoding a peptidelinker) to form a nucleic acid sequence encoding the chimeric receptor,using methods such as PCR amplification or ligation. Alternatively, thenucleic acid encoding the chimeric receptor may be synthesized. In someembodiments, the nucleic acid is DNA. In other embodiments, the nucleicacid is RNA.

Mutation of one or more residues within one or more of the components ofthe chimeric receptor (e.g., the antigen-binding fragment, etc.) may beperformed prior to or after joining the sequences of each of thecomponents. In some embodiments, one or more mutations in a component ofthe chimeric receptor may be made to modulate (increase or decrease) theaffinity of the component for an epitope (e.g., the antigen-bindingfragment for the target protein) and/or modulate the activity of thecomponent.

Any of the chimeric receptors described herein can be introduced into asuitable immune cell for expression via conventional technology. In someembodiments, the immune cells are T cells, such as primary T cells or Tcell lines. Alternatively, the immune cells can be NK cells, such asestablished NK cell lines (e.g., NK-92 cells). In some embodiments, theimmune cells are T cells that express CD8 (CD8⁺) or CD8 and CD4(CD8⁺/CD4⁺). In some embodiments, the T cells are T cells of anestablished T cell line, for example, 293T cells or Jurkat cells.

Primary T cells may be obtained from any source, such as peripheralblood mononuclear cells (PBMCs), bone marrow, tissues such as spleen,lymph node, thymus, or tumor tissue. A source suitable for obtaining thetype of immune cells desired would be evident to one of skill in theart. In some embodiments, the population of immune cells is derived froma human patient having a hematopoietic malignancy, such as from the bonemarrow or from PBMCs obtained from the patient. In some embodiments, thepopulation of immune cells is derived from a healthy donor. In someembodiments, the immune cells are obtained from the subject to whom theimmune cells expressing the chimeric receptors will be subsequentlyadministered. Immune cells that are administered to the same subjectfrom which the cells were obtained are referred to as autologous cells,whereas immune cells that are obtained from a subject who is not thesubject to whom the cells will be administered are referred to asallogeneic cells.

The type of host cells desired may be expanded within the population ofcells obtained by co-incubating the cells with stimulatory molecules,for example, anti-CD3 and anti-CD28 antibodies may be used for expansionof T cells.

To construct the immune cells that express any of the chimeric receptorconstructs described herein, expression vectors for stable or transientexpression of the chimeric receptor construct may be constructed viaconventional methods as described herein and introduced into immune hostcells. For example, nucleic acids encoding the chimeric receptors may becloned into a suitable expression vector, such as a viral vector inoperable linkage to a suitable promoter. The nucleic acids and thevector may be contacted, under suitable conditions, with a restrictionenzyme to create complementary ends on each molecule that can pair witheach other and be joined with a ligase. Alternatively, synthetic nucleicacid linkers can be ligated to the termini of the nucleic acid encodingthe chimeric receptors. The synthetic linkers may contain nucleic acidsequences that correspond to a particular restriction site in thevector. The selection of expression vectors/plasmids/viral vectors woulddepend on the type of host cells for expression of the chimericreceptors, but should be suitable for integration and replication ineukaryotic cells.

In some embodiments, the chimeric receptors are expressed using anon-integrating transient expression system. In some embodiments, thechimeric receptors are integrated into the genome of the immune cell. Insome embodiments, the chimeric receptors are integrated into a specificloci of the genome of immune cell using gene editing (e.g., zinc-fingernucleases, meganucleases, TALENs, CRISPR systems).

A variety of promoters can be used for expression of the chimericreceptors described herein, including, without limitation,cytomegalovirus (CMV) intermediate early promoter, a viral LTR such asthe Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemiavirus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleenfocus-forming virus (SFFV) LTR, the simian virus 40 (SV40) earlypromoter, herpes simplex tk virus promoter, elongation factor 1-alpha(EF1-α) promoter with or without the EF1-α intron. Additional promotersfor expression of the chimeric receptors include any constitutivelyactive promoter in an immune cell. Alternatively, any regulatablepromoter may be used, such that its expression can be modulated withinan immune cell. In some embodiments, the promoter regulating expressionof a chimeric receptor is an inducible promoter. In general, theactivity of “inducible promoters” may be regulated based on the presence(or absence) of a signal, such as an endogenous signal or an exogenoussignal, for example a small molecule or drug that is administered to thesubject.

Additionally, the vector may contain, for example, some or all of thefollowing: a selectable marker gene, such as the neomycin gene forselection of stable or transient transfectants in host cells;enhancer/promoter sequences from the immediate early gene of human CMVfor high levels of transcription; transcription termination and RNAprocessing signals from SV40 for mRNA stability; 5′- and 3′-untranslatedregions for mRNA stability and translation efficiency fromhighly-expressed genes like α-globin or β-globin; SV40 polyoma originsof replication and ColE1 for proper episomal replication; internalribosome binding sites (IRESes), versatile multiple cloning sites; T7and SP6 RNA promoters for in vitro transcription of sense and antisenseRNA; a “suicide switch” or “suicide gene” which when triggered causescells carrying the vector to die (e.g., HSV thymidine kinase, aninducible caspase such as iCasp9, drug-induced suicide switch,monoclonal antibody mediated suicide switch), and reporter gene forassessing expression of the chimeric receptor. Suitable vectors andmethods for producing vectors containing transgenes are well known andavailable in the art. Examples of the preparation of vectors forexpression of chimeric receptors can be found, for example, inUS2014/0106449, herein incorporated by reference in its entirety.

As will be appreciated by one of ordinary skill in the art, in someembodiments, it may be advantageous to quickly and efficiently reduce oreliminate the immune cells expressing chimeric receptors, for example ata time point following administration to a subject. Mechanisms ofkilling immune cells expressing chimeric receptors, or inducingcytotoxicity of such cells, are known in the art, see, e.g., Labanieh etal. Nature Biomedical Engineering (2018)2: 337-391; Slaney et al. CancerDiscovery (2018) 8(8): 924-934. In some embodiments, the immune cellexpressing the chimeric receptor may also express a suicide switch” or“suicide gene,” which may or may not be encoded by the same vector asthe chimeric receptor, as described above. In some embodiments, theimmune cell expressing chimeric receptors further express an epitope tagsuch that upon engagement of the epitope tag, the immune cell is killedthrough antibody-dependent cell-mediated cytotoxicity and/orcomplement-mediated cytotoxicity. See, e.g., Paszkiewicz et al. J. Clin.Invest. (2016) 126: 4262-4272; Wang et al. Blood (2011) 118: 1255-1263;Tasian et al. Blood (2017) 129: 2395-2407; Philip et al. Blood (2014)124: 1277-1287.

In some embodiments, the chimeric receptor construct or the nucleic acidencoding said chimeric receptor is a DNA molecule. In some embodiments,chimeric receptor construct or the nucleic acid encoding said chimericreceptor is a DNA vector and may be electroporated to immune cells (see,e.g., Till, et al. Blood (2012) 119(17): 3940-3950). In someembodiments, the nucleic acid encoding the chimeric receptor is an RNAmolecule, which may be electroporated to immune cells.

Any of the vectors comprising a nucleic acid sequence that encodes achimeric receptor construct described herein is also within the scope ofthe present disclosure. Such a vector may be delivered into host cellssuch as host immune cells by a suitable method. Methods of deliveringvectors to immune cells are well known in the art and may include DNA,RNA, or transposon electroporation, transfection reagents such asliposomes or nanoparticles to delivery DNA, RNA, or transposons;delivery of DNA, RNA, or transposons or protein by mechanicaldeformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013)110(6): 2082-2087); or viral transduction. In some embodiments, thevectors for expression of the chimeric receptors are delivered to hostcells by viral transduction. Exemplary viral methods for deliveryinclude, but are not limited to, recombinant retroviruses (see, e.g.,PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234;WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and4,777,127; GB Patent No. 2,200,651; and EP Patent No. 0 345 242),alphavirus-based vectors, and adeno-associated virus (AAV) vectors (see,e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO94/28938; WO 95/11984 and WO 95/00655). In some embodiments, the vectorsfor expression of the chimeric receptors are retroviruses. In someembodiments, the vectors for expression of the chimeric receptors arelentiviruses. In some embodiments, the vectors for expression of thechimeric receptors are adeno-associated viruses.

In examples in which the vectors encoding chimeric receptors areintroduced to the host cells using a viral vector, viral particles thatare capable of infecting the immune cells and carry the vector may beproduced by any method known in the art and can be found, for example inPCT Application No. WO 1991/002805A2, WO 1998/009271 A1, and U.S. Pat.No. 6,194,191. The viral particles are harvested from the cell culturesupernatant and may be isolated and/or purified prior to contacting theviral particles with the immune cells.

In some embodiments, the methods described herein involve use of immunecells that express more than one chimeric receptor (e.g., a chimericreceptors that target first epitope and chimeric receptors that target asecond epitope). In some embodiments, more than one chimeric receptor(e.g., a chimeric receptors that target first epitope and chimericreceptors that target a second epitope) are expressed from a singlevector. In some embodiments, more than one chimeric receptor (e.g., achimeric receptors that target first epitope and chimeric receptors thattarget a second epitope) are expressed from a more than one vector. Suchimmune cells may be prepared using methods known in the art, for exampleby delivering a vector that encodes a first chimeric receptorsimultaneously or sequentially with a second vector that encodes asecond chimeric receptor. In some embodiments, the resulting immune cellpopulation is a mixed population comprising a subset of immune cellsthat express one chimeric receptor and a subset of immune cells thatexpress both chimeric receptors.

In some embodiments, the domains of the chimeric receptor are encoded byand expressed from a single vector. Alternatively, the domains of thechimeric receptor may be encoded by and expressed from more than onevector. In some embodiments, activity of immune cells expressing thechimeric receptors may be regulated by controlling assembly of thechimeric receptor. The domains of a chimeric receptor may be expressedas two or more non-functional segments of the chimeric receptor andinduced to assemble at a desired time and/or location, for examplethrough use of a dimerization agent (e.g., dimerizing drug).

The methods of preparing host cells expressing any of the chimericreceptors described herein may comprise activating and/or expanding theimmune cells ex vivo. Activating a host cell means stimulating a hostcell into an activate state in which the cell may be able to performeffector functions (e.g., cytotoxicity). Methods of activating a hostcell will depend on the type of host cell used for expression of thechimeric receptors. Expanding host cells may involve any method thatresults in an increase in the number of cells expressing chimericreceptors, for example, allowing the host cells to proliferate orstimulating the host cells to proliferate. Methods for stimulatingexpansion of host cells will depend on the type of host cell used forexpression of the chimeric receptors and will be evident to one of skillin the art. In some embodiments, the host cells expressing any of thechimeric receptors described herein are activated and/or expanded exvivo prior to administration to a subject.

It has been appreciated that administration of immunostimulatorycytokines may enhance proliferation of immune cells expressing chimericreceptors following administration to a subject and/or promoteengraftment of the immune cells. See, e.g., Pegram et al. Leukemia(2015) 29:415-422; Chinnasamy et al. Clin. Cancer Res. (2012) 18:1672-1683; Krenciute et al. Cancer Immunol. Res. (2017) 5: 571-581; Huet al. Cell Rep. (2017) 20: 3025-3033; Markley et al. Blood (2010) 115:3508-3519). Any of the methods described herein may further involveadministering one or more immunostimulatory cytokines concurrently withany of the immune cells expressing chimeric receptors. Non-limitingexample of immunostimulatory cytokines include IL-12, IL-15, IL-1,IL-21, and combinations thereof.

Any anti-CD19 CAR and anti-CD33 CAR molecules known in the art can beused together with the genetically engineered HSCs described herein.Exemplary anti-CD19 CARs include axicabtagene and tisagenlecleucel.Exemplary anti-CD33 CARs include those disclosed in WO2017/066760 andWO2017/079400.

In one specific example, primary human CD8⁺ T cells are isolated frompatients' peripheral blood by immunomagnetic separation (MiltenyiBiotec®). T cells are cultured and stimulated with anti-CD3 andanti-CD28 mAbs-coated beads (Invitrogen®) as previously described(Levine et al., J. Immunol. (1997) 159(12):5921).

Chimeric receptors that target a lineage-specific cell-surface proteins(e.g., CD19 or CD33) are generated using conventional recombinant DNAtechnologies and inserted into a lentiviral vector. The vectorscontaining the chimeric receptors are used to generate lentiviralparticles, which are used to transduce primary CD8⁺ T cells. Humanrecombinant IL-2 may be added every other day (50 IU/mL). T cells arecultured for ˜14 days after stimulation. Expression of the chimericreceptors can be confirmed using methods, such as Western blotting andflow cytometry.

T cells expressing the chimeric receptors are selected and assessed fortheir ability to bind to the lineage-specific cell-surface protein suchas CD19 or CD33 and to induce cytotoxicity of cells expressing thelineage-specific protein. Immune cells expressing the chimeric receptorare also evaluated for their ability to induce cytotoxicity of cellsexpressing the lineage-specific antigen in mutated form. Preferably,immune cells expressing chimeric receptors that binds to the wild-typelineage-specific protein but not the mutated form. (FIG. 3, using CD33as an example).

The cells (e.g., hematopoietic stem cells) that express the mutatedlineage-specific protein are also assessed for various characteristics,including proliferation, erythropoietic differentiation, and colonyformation to confirm that the mutated lineage-specific protein retainedthe bioactivity as the wild-type counterpart.

The immune cells expressing one or more CAR-T receptors may be furthermodified genetically, for example, by knock-out of the native T-cellreceptor (TCR) or an MHC chain and/or by introducing a new TCR.Alternatively, the immune cells may retain the native TCR loci.

In some embodiments, the methods described herein involve administeringto the subject a population of genetically engineered cells lacking anon-essential epitope in a lineage-specific cell-surface antigen and oneor more immunotherapeutic agents (e.g., immune cells expressing chimericreceptor(s)) that target cells expressing the lineage-specificcell-surface antigen. In some embodiments, the methods described hereininvolve administering to the subject a population of geneticallyengineered cells lacking a non-essential epitope in a type 1lineage-specific cell-surface antigen and one or more immunotherapeuticagents (e.g., immune cells expressing chimeric receptor(s)) that targetcells expressing the lineage-specific cell-surface antigen. In someembodiments, the methods described herein involve administering to thesubject a population of genetically engineered cells lacking anon-essential epitope in a type 2 lineage-specific cell-surface antigenand one or more immunotherapeutic agents (e.g., immune cells expressingchimeric receptor(s)) that target cells expressing the lineage-specificcell-surface antigen. In any of the embodiments described herein, one ormore additional immunotherapeutic agents may be further administered tothe subject (e.g., targeting one or more additional epitopes and/orantigens), for example if the hematopoietic malignancy relapses.

In some embodiments, the methods described herein involve administeringimmune cells expressing chimeric receptors that target an epitope of alineage-specific cell-surface protein that is mutated in the populationof genetically engineered hematopoietic cells. In some embodiments, themethods described herein involve administering immune cells expressingchimeric receptors that target an epitope of a lineage-specificcell-surface protein that is mutated in the population of geneticallyengineered hematopoietic cells and one or more additional cytotoxicagents that may target one or more additional cell-surface proteins. Insome embodiments, the agents could work synergistically to enhanceefficacy by targeting more than one cell-surface protein.

In some examples, the methods described herein involve administering tothe subject a population of genetically engineered cells lacking anon-essential epitope of CD33 and one or more immune cells expressingchimeric receptor(s) that target cells expressing CD33. In someexamples, the methods described herein involve administering to thesubject a population of genetically engineered cells lacking an epitopein exon 2 or exon 3 of CD33 and one or more immune cells expressingchimeric receptor(s) that target cells expressing CD33. In someexamples, the methods described herein involve administering to thesubject a population of genetically engineered cells expressing amutated CD33 comprising the amino acid sequence of SEQ ID NO: 56 or SEQID NO: 58 and one or more immune cells expressing chimeric receptor(s)that target cells expressing CD33.

In some examples, the methods described herein involve administering tothe subject a population of genetically engineered cells lacking anon-essential epitope of CD19 and one or more immune cells expressingchimeric receptor(s) that target cells expressing CD19. In someexamples, the methods described herein involve administering to thesubject a population of genetically engineered cells lacking an epitopein exon 2 or exon 4 of CD19 and one or more immune cells expressingchimeric receptor(s) that target cells expressing CD19. In someexamples, the methods described herein involve administering to thesubject a population of genetically engineered cells expressing amutated CD19 comprising the amino acid sequence of SEQ ID NO: 52 or SEQID NO: 73 and one or more immune cells expressing chimeric receptor(s)that target cells expressing CD19.

(iii) Antibody-Drug Conjugate

In some embodiments, the cytotoxic agent targeting an epitope of acell-surface lineage-specific protein is an antibody-drug conjugate(ADC). As will be evident to one of ordinary skill in the art, the term“antibody-drug conjugate” can be used interchangeably with “immunotoxin”and refers to a fusion molecule comprising an antibody (orantigen-binding fragment thereof) conjugated to a toxin or drugmolecule. Binding of the antibody to the corresponding epitope of thetarget protein allows for delivery of the toxin or drug molecule to acell that presents the protein (and epitope thereof) on the cell surface(e.g., target cell), thereby resulting in death of the target cell. Insome embodiments, the antibody-drug conjugate (or antigen-bindingfragment thereof) binds to its corresponding epitope of alineage-specific cell-surface antigen but does not bind to alineage-specific cell-surface antigen that lacks the epitope or in whichthe epitope has been mutated.

In some embodiments, the agent is an antibody-drug conjugate. In someembodiments, the antibody-drug conjugate comprises an antigen-bindingfragment and a toxin or drug that induces cytotoxicity in a target cell.In some embodiments, the antibody-drug conjugate targets a type 2protein. In some embodiments, the antibody-drug conjugate targets CD33.In some embodiments, the antibody-drug conjugate binds to an epitope inexon 2 or exon 3 of CD33. In some embodiments, the antibody-drugconjugate targets a type 1 protein. In some embodiments, theantibody-drug conjugate binds to an epitope in exon 2 or exon 4 of CD19.In some embodiments, the antibody-drug conjugate targets CD19. In someembodiments, the antibody-drug conjugate targets a type 0 protein.

Toxins or drugs compatible for use in antibody-drug conjugates are wellknown in the art and will be evident to one of ordinary skill in theart. See, e.g., Peters et al. Biosci. Rep. (2015) 35(4): e00225, Beck etal. Nature Reviews Drug Discovery (2017) 16:315-337; Marin-Acevedo etal. J. Hematol. Oncol. (2018)11: 8; Elgundi et al. Advanced DrugDelivery Reviews (2017) 122: 2-19. In some embodiments, theantibody-drug conjugate may further comprise a linker (e.g., a peptidelinker, such as a cleavable linker or a non-cleavable linker) attachingthe antibody and drug molecule. Examples of antibody-drug conjugatesinclude, without limitation, brentuximab vedotin, glembatumumabvedotin/CDX-011, depatuxizumab mafodotin/ABT-414, PSMA ADC, polatuzumabvedotin/RG7596/DCDS4501A, denintuzumab mafodotin/SGN-CD19A, AGS-16C3F,CDX-014, RG7841/DLYE5953A, RG7882/DMUC406A, RG7986/DCDS0780A, SGN-LIV1A,enfortumab vedotin/ASG-22ME, AG-15ME, AGS67E, telisotuzumabvedotin/ABBV-399, ABBV-221, ABBV-085, GSK-2857916, tisotumabvedotin/HuMax®-TF-ADC, HuMax®-Ax1-ADC, pinatuzumabveodtin/RG7593/DCDT2980S, lifastuzumab vedotin/RG7599/DNIB0600A,indusatumab vedotin/MLN-0264/TAK-264, vandortuzumabvedotin/RG7450/DSTP3086S, sofituzumab vedotin/RG7458/DMUC5754A,RG7600/DMOT4039A, RG7336/DEDN6526A, ME1547, PF-06263507/ADC 5T4,trastuzumab emtansine/T-DM1, mirvetuximab soravtansine/IMGN853,coltuximab ravtansine/SAR3419, naratuximab emtansine/IMGN529,indatuximab ravtansine/BT-062, anetumab ravtansine/BAY 94-9343,SAR408701, SAR428926, AMG 224, PCA062, HKT288, LY3076226, SAR566658,lorvotuzumab mertansine/IMGN901, cantuzumab mertansine/SB-408075,cantuzumab ravtansine/IMGN242, laprituximab emtansine/IMGN289, IMGN388,bivatuzumab mertansine, AVE9633, BIIB015, MLN2704, AMG 172, AMG 595, LOP628, vadastuximab talirine/SGN-CD33A, SGN-CD70A, SGN-CD19B, SGN-CD123A,SGN-CD352A, rovalpituzumab tesirine/SC16LD6.5, SC-002, SC-003,ADCT-301/HuMax®-TAC-PBD, ADCT-402, MEDI3726/ADC-401, IMGN779, IMGN632,gemtuzumab ozogamicin, inotuzumab ozogamicin/CMC-544, PF-06647263,CMD-193, CMB-401, trastuzumab duocarmazine/SYD985, BMS-936561/MDX-1203,sacituzumab govitecan/IMMU-132, labetuzumab govitecan/IMMU-130,DS-8201a, U3-1402, milatuzumab doxorubicin/IMMU-110/hLL1-DOX,BMS-986148, RC48-ADC/hertuzumab-vc-MMAE, PF-06647020, PF-06650808,PF-06664178/RN927C, lupartumab amadotin/BAY1129980, aprutumabixadotin/BAY1187982, ARX788, AGS62P1, XMT-1522, AbGn-107, MEDI4276,DSTA4637S/RG7861. In one example, the antibody-drug conjugate isgemtuzumab ozogamicin.

In some embodiments, binding of the antibody-drug conjugate to theepitope of the cell-surface lineage-specific protein inducesinternalization of the antibody-drug conjugate, and the drug (or toxin)may be released intracellularly. In some embodiments, binding of theantibody-drug conjugate to the epitope of a cell-surfacelineage-specific protein induces internalization of the toxin or drug,which allows the toxin or drug to kill the cells expressing thelineage-specific protein (target cells). In some embodiments, binding ofthe antibody-drug conjugate to the epitope of a cell-surfacelineage-specific protein induces internalization of the toxin or drug,which may regulate the activity of the cell expressing thelineage-specific protein (target cells). The type of toxin or drug usedin the antibody-drug conjugates described herein is not limited to anyspecific type.

In some embodiments, two or more (e.g., 2, 3, 4, 5 or more) epitopes ofa lineage-specific cell-surface antigen have been modified, enabling twoor more (e.g., 2, 3, 4, 5 or more) different cytotoxic agents (e.g., twoADCs) to be targeted to the two or more epitopes. In some embodiments,the toxins carried by the ADCs could work synergistically to enhanceefficacy (e.g., death of the target cells). In some embodiments,epitopes of two or more (e.g., 2, 3, 4, 5 or more) lineage-specific cellsurface protein have been modified, enabling two or more (e.g., 2, 3, 4,5 or more) different cytotoxic agents (e.g., two ADCs) to be targeted toepitopes of the two or more lineage-specific cell-surface antigens. Insome embodiments, one or more (e.g., 1, 2, 3, 4, 5 or more) epitopes ofa lineage-specific cell-surface antigen have been modified and one ormore (e.g., 1, 2, 3, 4, 5 or more) epitopes of an additionalcell-surface protein have been modified, enabling two or more (e.g., 2,3, 4, 5 or more) different cytotoxic agents (e.g., two ADCs) to betargeted to epitopes of the lineage-specific cell-surface antigen andepitopes of additional cell-surface antigen. In some embodiments,targeting of more than one lineage-specific cell-surface antigen or alineage-specific cell-surface antigen and one or more additionalcell-surface protein/antigen may reduce relapse of a hematopoieticmalignancy.

In some embodiments, the methods described herein involve administeringADCs that target an epitope of a lineage-specific cell-surface antigenthat is mutated in the population of genetically engineeredhematopoietic cells. In some embodiments, the methods described hereininvolve administering ADCs that target an epitope of a lineage-specificcell-surface antigen that is mutated in the population of geneticallyengineered hematopoietic cells and one or more additional cytotoxicagents that may target one or more additional cell-surface proteins. Insome embodiments, the agents could work synergistically to enhanceefficacy by targeting more than one cell-surface protein.

An ADC described herein may be used as a follow-on treatment to subjectswho have been undergone the combined therapy as described herein.

In some embodiments, the methods described herein involve administeringto the subject a population of genetically engineered cells lacking anon-essential epitope in a lineage-specific cell-surface antigen and oneor more immunotherapeutic agents (e.g., ADCs) that target cellsexpressing the lineage-specific cell-surface antigen. In someembodiments, the methods described herein involve administering to thesubject a population of genetically engineered cells lacking anon-essential epitope in a type 1 lineage-specific cell-surface antigenand one or more immunotherapeutic agents (e.g., ADCs) that target cellsexpressing the lineage-specific cell-surface antigen. In someembodiments, the methods described herein involve administering to thesubject a population of genetically engineered cells lacking anon-essential epitope in a type 2 lineage-specific cell-surface antigenand one or more immunotherapeutic agents (e.g., ADCs) that target cellsexpressing the lineage-specific cell-surface antigen. In any of theembodiments described herein, one or more additional immunotherapeuticagents may be further administered to the subject (e.g., targeting oneor more additional epitopes and/or antigens), for example if thehematopoietic malignancy relapses.

In some examples, the methods described herein involve administering tothe subject a population of genetically engineered cells lacking anon-essential epitope of CD33 and one or more antibody-drug conjugatesthat target cells expressing CD33. In some examples, the methodsdescribed herein involve administering to the subject a population ofgenetically engineered cells lacking an epitope in exon 2 or exon 3 ofCD33 and one or more antibodies antibody-drug conjugates that targetcells expressing CD33. In some examples, the methods described hereininvolve administering to the subject a population of geneticallyengineered cells expressing a mutated CD33 comprising the amino acidsequence of SEQ ID NO: 56 or SEQ ID NO: 58 and one or more antibody-drugconjugates that target cells expressing CD33.

In some examples, the methods described herein involve administering tothe subject a population of genetically engineered cells lacking anon-essential epitope of CD19 and one or more antibody-drug conjugatesthat target cells expressing CD19. In some examples, the methodsdescribed herein involve administering to the subject a population ofgenetically engineered cells lacking an epitope in exon 2 or exon 4 ofCD19 and one or more antibody-drug conjugates that target cellsexpressing CD19. In some examples, the methods described herein involveadministering to the subject a population of genetically engineeredcells expressing a mutated CD19 comprising the amino acid sequence ofSEQ ID NO: 52 or SEQ ID NO: 73 and one or more antibody-drug conjugatesthat target cells expressing CD19.

III. Methods of Treatment and Combination Therapies

The genetically engineered hematopoietic cells such as HSCs may beadministered to a subject in need of the treatment, either taken aloneor in combination of one or more cytotoxic agents that target one ormore lineage-specific cell-surface antigens as described herein. Sincethe hematopoietic cells are genetically edited in the genes of the oneor more lineage-specific cell-surface antigens, the hematopoietic cellsand/or descendant cells thereof would express the one or morelineage-specific cell-surface antigens in mutated form (e.g., butfunctional) such that they can escape being targeted by the cytotoxicagents, for example, CAR-T cells.

Thus, the present disclosure provides methods for treating ahematopoietic malignancy, the method comprising administering to asubject in need thereof (i) a population of the genetically engineeredhematopoietic cells described herein, and optionally (ii) a cytotoxicagent such as CAR-T cells that target a lineage-specific cell-surfaceantigen, the gene of which is genetically edited in the hematopoieticcells such that the cytotoxic agent does not target hematopoietic cellsor descendant cells thereof. The administration of (i) and (ii) may beconcurrently or in any order. In some embodiments, the cytotoxic agentsand/or the hematopoietic cells may be mixed with a pharmaceuticallyacceptable carrier to form a pharmaceutical composition, which is alsowithin the scope of the present disclosure.

In some embodiments, the genetically engineered hematopoietic stem cellshave genetic editing in genes of at least two lineage-specificcell-surface proteins/antigens A and B. Such hematopoietic stem cellscan be administered to a subject, who has been or will be treated with afirst cytotoxic agent specific to A (e.g., anti-protein A CAR-T cells),or who is at risk of a hematopoietic malignancy that would needtreatment of the cytotoxic agent. When the subject developed resistanceto the cytotoxic agent after the treatment or has relapse of thehematopoietic malignancy, a second cytotoxic agent specific to B (e.g.,anti-protein B CAR-T cells) may be administered to the subject. Becausethe genetically engineered hematopoietic cells have genetic editing inboth A and B genes, those cells and descendant cells thereof would alsobe resistant to cytotoxicity induced by the second cytotoxic agent. Assuch, administering once the genetically engineered hematopoietic cellswould be sufficient to compensate loss of normal cells expressing atleast lineage-specific cell-surface proteins/antigens A and B due tomultiple treatment by cytotoxic agents specific to at leastproteins/antigens A and B.

As used herein, “subject,” “individual,” and “patient” are usedinterchangeably, and refer to a vertebrate, preferably a mammal such asa human. Mammals include, but are not limited to, human primates,non-human primates or murine, bovine, equine, canine or feline species.In some embodiments, the subject is a human patient having ahematopoietic malignancy.

To perform the methods described herein, an effective amount of thegenetically engineered hematopoietic cells can be administered to asubject in need of the treatment. Optionally, the hematopoietic cellscan be co-used with a cytotoxic agent as described herein. As usedherein the term “effective amount” may be used interchangeably with theterm “therapeutically effective amount” and refers to that quantity of acytotoxic agent, hematopoietic cell population, or pharmaceuticalcomposition (e.g., a composition comprising cytotoxic agents and/orhematopoietic cells) that is sufficient to result in a desired activityupon administration to a subject in need thereof. Within the context ofthe present disclosure, the term “effective amount” refers to thatquantity of a compound, cell population, or pharmaceutical compositionthat is sufficient to delay the manifestation, arrest the progression,relieve or alleviate at least one symptom of a disorder treated by themethods of the present disclosure. Note that when a combination ofactive ingredients is administered the effective amount of thecombination may or may not include amounts of each ingredient that wouldhave been effective if administered individually.

Effective amounts vary, as recognized by those skilled in the art,depending on the particular condition being treated, the severity of thecondition, the individual patient parameters including age, physicalcondition, size, gender and weight, the duration of the treatment, thenature of concurrent therapy (if any), the specific route ofadministration and like factors within the knowledge and expertise ofthe health practitioner. In some embodiments, the effective amountalleviates, relieves, ameliorates, improves, reduces the symptoms, ordelays the progression of any disease or disorder in the subject. Insome embodiments, the subject is a human. In some embodiments, thesubject is a human patient having a hematopoietic malignancy.

As described herein, the hematopoietic cells and/or immune cellsexpressing chimeric receptors may be autologous to the subject, i.e.,the cells are obtained from the subject in need of the treatment,manipulated such that the cells do not bind the cytotoxic agents, andthen administered to the same subject. Administration of autologouscells to a subject may result in reduced rejection of the host cells ascompared to administration of non-autologous cells. Alternatively, thehost cells are allogeneic cells, i.e., the cells are obtained from afirst subject, manipulated such that the cells do not bind the cytotoxicagents, and then administered to a second subject that is different fromthe first subject but of the same species. For example, allogeneicimmune cells may be derived from a human donor and administered to ahuman recipient who is different from the donor.

In some embodiments, engineered hematopoietic cells comprising one ormore genetically engineered gene(s) encoding lineage-specificcell-surface protein(s) (e.g., CD33 or CD19 or another lineage-specificcell-surface protein described herein) and the immune cells engineeredto target an epitope of the lineage-specific cell-surface protein(s) areboth allogeneic to the subject. In some embodiments, engineeredhematopoietic cells comprising one or more genetically engineeredgene(s) encoding lineage-specific cell-surface protein(s) (e.g., CD33 orCD19 or another lineage-specific cell-surface protein described herein)and the immune cells engineered to target an epitope of thelineage-specific cell-surface protein(s) are from the same allogeneicdonor. In some embodiments, engineered hematopoietic cells comprisingone or more genetically engineered gene(s) encoding lineage-specificcell-surface protein(s) (e.g., CD33 or CD19 or another lineage-specificcell-surface protein described herein) and the immune cells engineeredto target an epitope of the lineage-specific cell-surface protein(s) arefrom two different allogeneic donors.

In some embodiments, engineered hematopoietic cells comprising one ormore genetically engineered gene(s) encoding lineage-specificcell-surface protein(s) (e.g., CD33 or CD19 or another lineage-specificcell-surface protein described herein) and the immune cells engineeredto target an epitope of the lineage-specific cell-surface protein(s) areboth autologous to the subject.

In some embodiments, engineered hematopoietic cells comprising one ormore genetically engineered gene(s) encoding lineage-specificcell-surface protein(s) (e.g., CD33 or CD19 or another lineage-specificcell-surface protein described herein) are autologous to the subject andthe immune cells engineered to target an epitope of the lineage-specificcell-surface protein(s) are from an allogeneic donor. In someembodiments, engineered hematopoietic cells comprising one or moregenetically engineered gene(s) encoding lineage-specific cell-surfaceprotein(s) (e.g., CD33 or CD19 or another lineage-specific cell-surfaceprotein described herein) are from an allogeneic donor and the immunecells engineered to target an epitope of the lineage-specificcell-surface protein(s) are autologous to the subject.

In some embodiments, the immune cells and/or hematopoietic cells areallogeneic cells and have been further genetically engineered to reducegraft-versus-host disease. For example, as described herein, thehematopoietic stem cells may be genetically engineered (e.g., usinggenome editing) to have reduced expression of CD45RA. Methods forreducing graft-versus-host disease are known in the art, see, e.g., Yanget al. Curr. Opin. Hematol. (2015) 22(6): 509-515. In some embodiments,the immune cells (e.g., T cells) may be genetically engineered to reduceor eliminate expression of the T cell receptor (TCR) or reduce oreliminate cell surface localization of the TCR. In some examples, thegene encoding the TCR is knocked out or silenced (e.g., using geneediting methods, or shRNAs). In some embodiments, the TCR is silencedusing peptide inhibitors of the TCR. In some embodiments, the immunecells (e.g., T cells) are subjected to a selection process to select forimmune cells or a population of immune cells that do not contain analloreactive TCR. Alternatively, in some embodiments, immune cells thatnaturally do not express TCRs (e.g., NK cells) may be used in any of themethods described herein.

In some embodiments, the immune cells and/or hematopoietic cells havebeen further genetically engineered to reduce host-versus-graft effects.For example, in some embodiments, immune cells and/or hematopoieticcells may be subjected to gene editing or silencing methods to reduce oreliminate expression of one or more proteins involved in inducing hostimmune responses, e.g., CD52, MHC molecules, and/or MHC beta-2microglobulin.

In some embodiments, the immune cells expressing any of the chimericreceptors described herein are administered to a subject in an amounteffective in to reduce the number of target cells (e.g., cancer cells)by least 20%, e.g., 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold,50-fold, 100-fold or more.

A typical amount of cells, i.e., immune cells or hematopoietic cells,administered to a mammal (e.g., a human) can be, for example, in therange of about 10⁶ to 10¹¹ cells. In some embodiments it may bedesirable to administer fewer than 10⁶ cells to the subject. In someembodiments, it may be desirable to administer more than 10¹¹ cells tothe subject. In some embodiments, one or more doses of cells includesabout 10⁶ cells to about 10¹¹ cells, about 10⁷ cells to about 10¹⁰cells, about 10⁸ cells to about 10⁹ cells, about 10⁶ cells to about 10⁸cells, about 10⁷ cells to about 10⁹ cells, about 10⁷ cells to about 10¹⁰cells, about 10⁷ cells to about 10¹¹ cells, about 10⁸ cells to about10¹⁰ cells, about 10⁸ cells to about 10¹¹ cells, about 10⁹ cells toabout 10¹⁰ cells, about 10⁹ cells to about 10¹¹ cells, or about 10¹⁰cells to about 10¹¹ cells.

In some embodiments, the subject is preconditioned prior toadministration of the cytotoxic agent and/or hematopoietic cells. Insome embodiments, the method further comprises pre-conditioning thesubject. In general, preconditioning a subject involves subjecting thepatient to one or more therapy, such as a chemotherapy or other type oftherapy, such as irradiation. In some embodiments, the preconditioningmay induce or enhance the patient's tolerance of one or more subsequenttherapy (e.g., a targeted therapy), as described herein. In someembodiments, the pre-conditioning involves administering one or morechemotherapeutic agents to the subject. Non-limiting examples ofchemotherapeutic agents include actinomycin, azacitidine, azathioprine,bleomycin, bortezomib, carboplatin, capecitabine, cisplatin,chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel,doxifluridine, doxorubicin, epirubicin, epothilone, etoposide,fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin,imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate,mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide,tioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine,and vinorelbine.

In some embodiments, the subject is preconditioned at least one day, twodays, three days, four days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, two weeks, three weeks, four weeks,five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks,two months, three months, four months, five months, or at least sixmonths prior to administering the cytotoxic agent and/or hematopoieticcells.

In other embodiments, the chemotherapy(ies) or other therapy(ies) areadministered concurrently with the cytotoxic agent and manipulatedhematopoietic cells. In other embodiments, the chemotherapy(ies) orother therapy(ies) are administered after the cytotoxic agent andmanipulated hematopoietic cells.

In one embodiment, the chimeric receptor (e.g., a nucleic acid encodingthe chimeric receptor) is introduced into an immune cell, and thesubject (e.g., human patient) receives an initial administration or doseof the immune cells expressing the chimeric receptor. One or moresubsequent administrations of the cytotoxic agent (e.g., immune cellsexpressing the chimeric receptor) may be provided to the patient atintervals of 15 days, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 daysafter the previous administration. More than one dose of the cytotoxicagent can be administered to the subject per week, e.g., 2, 3, 4, ormore administrations of the agent. The subject may receive more than onedoses of the cytotoxic agent (e.g., an immune cell expressing a chimericreceptor) per week, followed by a week of no administration of theagent, and finally followed by one or more additional doses of thecytotoxic agent (e.g., more than one administration of immune cellsexpressing a chimeric receptor per week). The immune cells expressing achimeric receptor may be administered every other day for 3administrations per week for two, three, four, five, six, seven, eightor more weeks.

Any of the methods described herein may be for the treatment of ahematological malignancy in a subject. As used herein, the terms“treat,” “treating,” and “treatment” mean to relieve or alleviate atleast one symptom associated with the disease or disorder, or to slow orreverse the progression of the disease or disorder. Within the meaningof the present disclosure, the term “treat” also denotes to arrest,delay the onset (i.e., the period prior to clinical manifestation of adisease) and/or reduce the risk of developing or worsening a disease.For example, in connection with cancer, the term “treat” may meaneliminate or reduce the number or replication of cancer cells, and/orprevent, delay or inhibit metastasis, etc.

In some embodiments, a population of genetically engineeredhematopoietic cells (e.g., carrying genetic edits in genes of one ormore lineage-specific cell-surface proteins for expressing thoseproteins in mutated form) and a cytotoxic agent(s) specific to thelineage-specific cell-surface protein are co-administered to a subjectvia a suitable route (e.g., infusion). In such a combined therapeuticmethods, the cytotoxic agent recognizes (binds) a target cell expressingthe cell-surface lineage-specific protein for targeted killing. Thehematopoietic cells that express the protein in mutated form, which hasreduced binding activity or do not bind the cytotoxic acid (e.g.,because of lacking binding epitope) allow for repopulation of a celltype that is targeted by the cytotoxic agent.

In some embodiments, the methods described herein involve administeringa population of genetically engineered hematopoietic cells to a subjectand administering one or more immunotherapeutic agents (e.g., cytotoxicagents). As will be appreciated by one of ordinary skill in the art, theimmunotherapeutic agents may be of the same or different type (e.g.,therapeutic antibodies, populations of immune cells expressing chimericantigen receptor(s), and/or antibody-drug conjugates).

In some embodiments, the methods described herein involve administeringa population of genetically engineered hematopoietic cells in which CD33is mutated to a subject and administering one or immunotherapeuticagents (e.g., cytotoxic agents). In some embodiments, the methodsdescribed herein involve administering a population of geneticallyengineered hematopoietic cells in which CD33 is mutated to a subject andadministering one or more therapeutic antibodies. In some embodiments,the methods described herein involve administering a population ofgenetically engineered hematopoietic cells in which CD33 is mutated to asubject and administering one or more populations of immune cellsexpressing chimeric antigen receptor(s). In some embodiments, themethods described herein involve administering a population ofgenetically engineered hematopoietic cells in which CD33 is mutated to asubject and administering one or more antibody-drug conjugates. In someembodiments, the methods described herein involve administering apopulation of genetically engineered hematopoietic cells comprising amutated CD33 set forth by SEQ ID NO: 56 or 58 to a subject andadministering one or more antibody-drug conjugates.

In some embodiments, the methods described herein involve administeringa population of genetically engineered hematopoietic cells in which CD19is mutated to a subject and administering one or immunotherapeuticagents (e.g., cytotoxic agents). In some embodiments, the methodsdescribed herein involve administering a population of geneticallyengineered hematopoietic cells in which CD19 is mutated to a subject andadministering one or more therapeutic antibodies. In some embodiments,the methods described herein involve administering a population ofgenetically engineered hematopoietic cells in which CD19 is mutated to asubject and administering one or more populations of immune cellsexpressing chimeric antigen receptor(s). In some embodiments, themethods described herein involve administering a population ofgenetically engineered hematopoietic cells in which CD19 is mutated to asubject and administering one or more antibody-drug conjugates. In someembodiments, the methods described herein involve administering apopulation of genetically engineered hematopoietic cells comprising amutated CD19 set forth by SEQ ID NO: 52 or 73 to a subject andadministering one or more antibody-drug conjugates.

In some embodiments, the treatment of the patient can involve thefollowing steps: (1) administering a therapeutically effective amount ofthe cytotoxic agent to the patient and (2) infusing or reinfusing thepatient with hematopoietic stem cells, either autologous or allogenic.In some embodiments, the treatment of the patient can involve thefollowing steps: (1) administering a therapeutically effective amount ofan immune cell expressing a chimeric receptor to the patient, whereinthe immune cell comprises a nucleic acid sequence encoding a chimericreceptor that binds an epitope of a cell-surface lineage-specific,disease-associated protein; and (2) infusing or reinfusing the patientwith hematopoietic cells (e.g., hematopoietic stem cells), eitherautologous or allogenic. In each of the methods described herein, thecytotoxic agent (e.g., CAR-T cells) and the genetically engineeredhematopoietic cells can be administered to the subject in any order. Insome instances, the hematopoietic cells are given to the subject priorto the cytotoxic agent. In some instances, a second cytotoxic agent canbe administered to the subject after treatment with the first cytotoxicagent, e.g., when the patient develops resistance or disease relapse.The hematopoietic cells given the same subject may have multiple editedgenes expressing lineage-specific cell-surface proteins in mutated formsuch that the cytotoxic agents can target wild-type proteins but not themutated form.

The efficacy of the therapeutic methods using a population ofgenetically engineered hematopoietic cells described herein, optionallyin combination with a cytotoxic agent (e.g., CART) may be assessed byany method known in the art and would be evident to a skilled medicalprofessional. For example, the efficacy of the therapy may be assessedby survival of the subject or cancer burden in the subject or tissue orsample thereof. In some embodiments, the efficacy of the therapy isassessed by quantifying the number of cells belonging to a particularpopulation or lineage of cells. In some embodiments, the efficacy of thetherapy is assessed by quantifying the number of cells presenting thecell-surface lineage-specific protein.

In some embodiments, the cytotoxic agent comprising an antigen-bindingfragment that binds to the epitope of the cell-surface lineage-specificprotein and the population of hematopoietic cells is administeredconcomitantly.

In some embodiments, the cytotoxic agent comprising an antigen-bindingfragment that binds an epitope of a cell-surface lineage-specificprotein (e.g., immune cells expressing a chimeric receptor as describedherein) is administered prior to administration of the hematopoieticcells. In some embodiments, the agent comprising an antigen-bindingfragment that binds an epitope of a cell-surface lineage-specificprotein (e.g., immune cells expressing a chimeric receptor as describedherein) is administered at least about 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4months, 5 months, 6 months or more prior to administration of thehematopoietic cells.

In some embodiments, the hematopoietic cells are administered prior tothe cytotoxic agent comprising an antigen-binding fragment that binds anepitope of the cell-surface lineage-specific protein (e.g., immune cellsexpressing a chimeric receptor as described herein). In someembodiments, the population of hematopoietic cells is administered atleast about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months ormore prior to administration of the cytotoxic agent comprising anantigen-binding fragment that binds to an epitope of the cell-surfacelineage-specific protein.

In some embodiments, the cytotoxic agent targeting the cell-surfacelineage-specific protein and the population of hematopoietic cells areadministered at substantially the same time. In some embodiments, thecytotoxic agent targeting the cell-surface lineage-specific protein isadministered and the patient is assessed for a period of time, afterwhich the population of hematopoietic cells is administered. In someembodiments, the population of hematopoietic cells is administered andthe patient is assessed for a period of time, after which the cytotoxicagent targeting the cell-surface lineage-specific protein isadministered.

Also within the scope of the present disclosure are multipleadministrations (e.g., doses) of the cytotoxic agents and/or populationsof hematopoietic cells. In some embodiments, the cytotoxic agents and/orpopulations of hematopoietic cells are administered to the subject once.In some embodiments, cytotoxic agents and/or populations ofhematopoietic cells are administered to the subject more than once(e.g., at least 2, 3, 4, 5, or more times). In some embodiments, thecytotoxic agents and/or populations of hematopoietic cells areadministered to the subject at a regular interval, e.g., every sixmonths.

In some embodiments, the subject is a human subject having ahematopoietic malignancy. As used herein a hematopoietic malignancyrefers to a malignant abnormality involving hematopoietic cells (e.g.,blood cells, including progenitor and stem cells). Examples ofhematopoietic malignancies include, without limitation, Hodgkin'slymphoma, non-Hodgkin's lymphoma, leukemia, or multiple myeloma.Exemplary leukemias include, without limitation, acute myeloid leukemia,acute lymphoid leukemia, chronic myelogenous leukemia, acutelymphoblastic leukemia or chronic lymphoblastic leukemia, and chroniclymphoid leukemia.

In some embodiments, cells involved in the hematopoietic malignancy areresistant to convention or standard therapeutics used to treat themalignancy. For example, the cells (e.g., cancer cells) may be resistantto a chemotherapeutic agent and/or CAR T cells used to treat themalignancy.

In some embodiments, the hematopoietic malignancy is associated withCD19⁺ cells. Examples include, but are not limited to, B cellmalignancies such as non-Hodgkin's lymphoma, Hodgkin's lymphoma,leukemia, multiple myeloma, acute lymphoblastic leukemia, acute lymphoidleukemia, acute lymphocytic leukemia, chronic lymphoblastic leukemia,chronic lymphoid leukemia, and chronic lymphocytic leukemia. In someembodiments, the hematopoietic malignancy is a relapsing hematopoieticmalignancy.

In some embodiments, the leukemia is acute myeloid leukemia (AML). AMLis characterized as a heterogeneous, clonal, neoplastic disease thatoriginates from transformed cells that have progressively acquiredcritical genetic changes that disrupt key differentiation andgrowth-regulatory pathways. (Dohner et al., NEJM, (2015) 373:1136). CD33glycoprotein is expressed on the majority of myeloid leukemia cells aswell as on normal myeloid and monocytic precursors and has beenconsidered to be an attractive target for AML therapy (Laszlo et al.,Blood Rev. (2014) 28(4):143-53). While clinical trials using anti-CD33monoclonal antibody based therapy have shown improved survival in asubset of AML patients when combined with standard chemotherapy, theseeffects were also accompanied by safety and efficacy concerns.

In some cases, a subject may initially respond to a therapy (e.g., for ahematopoietic malignancy) and subsequently experience relapse. Any ofthe methods or populations of genetically engineered hematopoietic cellsdescribed herein may be used to reduce or prevent relapse of ahematopoietic malignancy. Alternatively or in addition, any of themethods described herein may involve administering any of thepopulations of genetically engineered hematopoietic cells describedherein and an immunotherapeutic agent (e.g., cytotoxic agent) thattargets cells associated with the hematopoietic malignancy and furtheradministering one or more additional immunotherapeutic agents when thehematopoietic malignancy relapses.

As used herein, the term “relapse” refers to the reemergence orreappearance of cells associated with a hematopoietic malignancyfollowing a period of responsiveness to a therapy. Methods ofdetermining whether a hematopoietic malignancy has relapsed in a subjectwill appreciated by one of ordinary skill in the art. In someembodiments, the period of responsiveness to a therapy involves thelevel or quantity of cells associated with the hematopoietic malignancythe falling below a threshold, e.g., below 20%, 15%, 10%, 5%, 4%, 3%,2%, or 1% of the level or quantity of cells prior to administration ofthe therapy. In some embodiments, a relapse is characterized by thelevel or quantity of cells associated with the hematopoietic malignancyabove a threshold, e.g., above 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%higher than the level or quantity of cells during the period ofresponsiveness. Methods of determining the minimal residual disease in asubject are known in the art and may be used, for example to assesswhether a hematopoietic malignancy has relapsed or is likely to relapse.See, e.g., Taraseviciute et al. Hematology and Oncology (2019) 31(1)).

In some embodiments, the subject has or is susceptible to relapse of ahematopoietic malignancy (e.g., AML) following administration of one ormore previous therapies. In some embodiments, the methods describedherein reduce the subject's risk of relapse or the severity of relapse.

Without wishing to be bound by any particular theory, some cancers,including hematopoietic malignancies, are thought to relapse after aninitial period of responsiveness to a therapy due to mechanisms such asantigen loss/antigen escape or lineage switch. In general, antigenloss/antigen escape results in relapse with a phenotypically similarhematopoietic malignancy characterized by cells that lack surfaceexpression of the antigen targeted by the previous therapy (e.g.,immunotherapeutic agent) such that the cells are no longer targeted bythe previous therapy. In contrast, lineage switch presents as agenetically related but phenotypically different malignancy in which thecells lack surface expression of the antigen targeted by the previoustherapy (e.g., immunotherapeutic agent) such that the cells are nolonger targeted by the previous therapy. See, e.g., Brown et al. NatureReviews Immunology (2019) 19:73-74; Majzner et al. Cancer Discovery(2018) 8(10).

Antigen loss/antigen escape in which the target antigen is no longerpresent on the target cells (e.g., cells of the hematopoieticmalignancy) frequently occurs as a result of genetic mutation and/orenrichment of cells that express a variant of the antigen (e.g.,lineage-specific cell-surface antigen) that is not targeted by theimmunotherapeutic agent (e.g., cytotoxic agent). In some embodiments,the hematopoietic malignancy has relapsed due to antigen loss/antigenescape. In some embodiments, the target cells have lost the targetedepitope (e.g., of a lineage-specific cell-surface antigen) or havereduced expression of the antigen (e.g., lineage-specific cell-surfaceantigen) such that the targeted epitope is not recognized by theimmunotherapeutic agent or is not sufficient to induce cytotoxicity. Insome embodiments, the hematopoietic malignancy has relapsed due tolineage switch.

In some embodiments, the methods described herein reduce or avoidrelapse of a hematopoietic malignancy by targeting more than one antigen(e.g., more than one lineage-specific cell-surface antigen). In someembodiments, the populations of genetically engineered hematopoieticcells express mutants of more than one lineage-specific surface antigenssuch that the mutated lineage-specific surface antigens are not targetedby an immunotherapeutic agent(s).

In some embodiments, a cancer treated with the methods herein comprisesa first sub-population of cancer cells and a second sub-population ofcancer cells. One of the sub-populations may be cancer stem cells. Oneof the sub-populations may be cancer bulk cells. One of thesub-populations may have one or more (e.g., at least 2, 3, 4, 5, or all)markers of differentiated hematopoietic cells. One of thesub-populations may have one or more (e.g., at least 2, 3, 4, 5, or all)markers of earlier lineage cells, e.g., HSCs or HPCs.

Markers characteristic of different sub-populations of cancer cells(e.g., cancer stem cells and cancer bulk cells) are described, e.g., invan Galen et al. Cell 176, 1-17, Mar. 7, 2019, which is hereinincorporated by reference in its entirety. For instance, in someembodiments, the first sub-population of cancer cells comprisesprimitive AML cells and/or the second sub-population of cancer cellscomprises differentiated AML cells (e.g., differentiated monocyte-likeAML cells). In some embodiments, a primitive AML cells expressesstemness genes (e.g., as described in van Galen et al., supra) and/ormyeloid priming genes (e.g., as described in van Galen et al., supra).In some embodiments, a differentiated monocyte-like AML cell expressesimmunomodulatory genes (e.g., as described in van Galen et al., supra).In some embodiments, one or more of the sub-populations of cancer cellsare chosen from: HSC-like, progenitor-like, GMP-like, promonocyte-like,monocyte-like, or conventional dendritic cell (cDC)-like, e.g., asdescribed in van Galen et al., supra.

Any of the immune cells expressing chimeric receptors described hereinmay be administered in a pharmaceutically acceptable carrier orexcipient as a pharmaceutical composition.

The phrase “pharmaceutically acceptable,” as used in connection withcompositions and/or cells of the present disclosure, refers to molecularentities and other ingredients of such compositions that arephysiologically tolerable and do not typically produce untowardreactions when administered to a mammal (e.g., a human). Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inmammals, and more particularly in humans. “Acceptable” means that thecarrier is compatible with the active ingredient of the composition(e.g., the nucleic acids, vectors, cells, or therapeutic antibodies) anddoes not negatively affect the subject to which the composition(s) areadministered. Any of the pharmaceutical compositions and/or cells to beused in the present methods can comprise pharmaceutically acceptablecarriers, excipients, or stabilizers in the form of lyophilizedformations or aqueous solutions.

Pharmaceutically acceptable carriers, including buffers, are well knownin the art, and may comprise phosphate, citrate, and other organicacids; antioxidants including ascorbic acid and methionine;preservatives; low molecular weight polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; amino acids; hydrophobicpolymers; monosaccharides; disaccharides; and other carbohydrates; metalcomplexes; and/or non-ionic surfactants. See, e.g. Remington: TheScience and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams andWilkins, Ed. K. E. Hoover.

Kits for Therapeutic Uses

Also within the scope of the present disclosure are kits for use intreating hematopoietic malignancy. Such a kit may comprise thegenetically engineered hematopoietic cells such as HSCs, and optionallyone or more cytotoxic agents targeting lineage-specific cell-surfaceantigens, the genes of which are edited in the hematopoietic cells. Suchkits may include a container comprising a first pharmaceuticalcomposition that comprises any of the genetically engineeredhematopoietic cells as described herein, and optionally one or moreadditional containers comprising one or more cytotoxic agents (e.g.,immune cells expressing chimeric receptors described herein) targetingthe lineage-specific cell-surface antigens as also described herein.

In some embodiments, the kit can comprise instructions for use in any ofthe methods described herein. The included instructions can comprise adescription of administration of the genetically engineeredhematopoietic cells and optionally descriptions of administration of theone or more cytotoxic agents to a subject to achieve the intendedactivity in a subject. The kit may further comprise a description ofselecting a subject suitable for treatment based on identifying whetherthe subject is in need of the treatment. In some embodiments, theinstructions comprise a description of administering the geneticallyengineered hematopoietic cells and optionally the one or more cytotoxicagents to a subject who is in need of the treatment.

The instructions relating to the use of the genetically engineeredhematopoietic cells and optionally the cytotoxic agents described hereingenerally include information as to dosage, dosing schedule, and routeof administration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the disclosure are typicallywritten instructions on a label or package insert. The label or packageinsert indicates that the pharmaceutical compositions are used fortreating, delaying the onset, and/or alleviating a disease or disorderin a subject.

The kits provided herein are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging, and the like. Also contemplated are packages for use incombination with a specific device, such as an inhaler, nasaladministration device, or an infusion device. A kit may have a sterileaccess port (for example, the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The container may also have a sterile access port. At least oneactive agent in the pharmaceutical composition is a chimeric receptorvariants as described herein.

Kits optionally may provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. In someembodiment, the disclosure provides articles of manufacture comprisingcontents of the kits described above.

General Techniques

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as Molecular Cloning: ALaboratory Manual, second edition (Sambrook, et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I.Freshney, ed. 1987); Introuction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.);Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell,eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P.Calos, eds., 1987); Current Protocols in Molecular Biology (F. M.Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis,et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan etal., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons,1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies(P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal antibodies: a practical approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); Usingantibodies: a laboratory manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practicalApproach, Volumes I and II (D. N. Glover ed. 1985); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. (1985»; Transcriptionand Translation (B. D. Hames & S. J. Higgins, eds. (1984»; Animal CellCulture (R. I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (IRLPress, (1986»; and B. Perbal, A practical Guide To Molecular Cloning(1984); F. M. Ausubel et al. (eds.).

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

Example 1: Deletion Exon 2 of CD19 Via CRISPR/Cas9-Mediated Gene Editingand Characterization of Cells Expressing Exon 2-Deleted CD19

This Example reports genetic engineering of CD19 genes via CRISPR/Cas9in cells to produce edited cells expressing mutated CD19, in which thefragment encoded by exon 2 is deleted (CD19ex2), and in vitro and invivo characterization of such edited cells. See also above disclosuresfor exemplary exon-2 deleted CD19 gene products.

Materials and Methods

Design of sgRNA Constructs

All sgRNAs were designed by manual inspection for the SpCas9 PAM(5′-NGG-3′) with close proximity to the target region and prioritizedaccording to predicted specificity by minimizing potential off-targetsites in the human genome with an online search algorithm (Benchling,Doench et al 2016, Hsu et al 2013). All designed synthetic sgRNAs werepurchased from Synthego® with chemically modified nucleotides at thethree terminal positions at both the 5′ and 3′ ends. Modifiednucleotides contained 2′-O-methyl-3′-phosphorothioate (abbreviated as“ms”) and the ms-sgRNAs were HPLC-purified. Cas9 protein was purchasedfrom Synthego® (FIGS. 5-8B) and Aldervon® (FIGS. 9A, 9B, 10A-10D,14A-14D, 17, 18A and 18B).

Cell Maintenance and Electroporation of Immortalized Human Cell Lines

K562 human leukemia cell lines were obtained from American Type CultureCollection® (ATCC®) and maintained in DMEM+10% FBS and maintained at 37°C. at 5% CO2. K562 cells were edited by electroporation of the Cas9ribonucleoprotein (RNP) using the Lonza® Nucleofector™ (program SF-220)and the Human P3 Cell Nucleofection Kit (VPA-1002, Lonza®).Raji-Fluc-GFP cells were purchased from Capital Biosciences™ andmaintained in RPMI+10% FBS+1% Glutamine at 37° C. at 5% CO2.Raji-Fluc-GFP cells were edited by electroporation of RNP using theLonza® Nucleofector™ (program DS-104) and SG Cell line 4D-Nucleofector™X Kit S (V4XC-3032, Lonza®). Cas9 RNP was made by incubating proteinwith ms-sgRNA at a molar ratio of 1:9 (20:180 pmol) at 25° C. for 10minutes immediately before electroporation. After electroporation, cellswere incubated for 10 minutes in the cuvette, transferred to 1 mL of theabove medium, and cultured for 24-72 hrs for downstream analysis.

Editing in Primary Human CD34+ HSCs

Frozen CD34+ HSCs derived from mobilized peripheral blood were purchasedfrom AllCells® and thawed according to manufacturer's instructions.Frozen CD34+ HSCs derived from cord blood were either purchased frozenfrom AllCells® or Stemcell™ and thawed and maintained according tomanufacturer's instructions. To edit HSCs, ˜1e6 HSCs were thawed andcultured in StemSpan™ SFEM medium supplemented with StemSpan™ CC110cocktail (StemCell Technologies™) for 24 h before electroporation withRNP. To electroporate HSCs, 1.5e5 were pelleted and resuspended in 20 μLLonza® P3 solution, and mixed with 10 uL Cas9 RNP as described above.CD34+ HSCs were electroporated using the Lonza® Nucleofector™ 2 (programDU-100) and the Human P3 Cell Nucleofection Kit (VPA-1002, Lonza®).

Genomic DNA Analysis

For all genomic analysis, DNA was harvested from cells using the Qiagen®DNeasy® kit. For T7E1 assays, PCR was performed with primers flankingthe CRISPR cut sites. Products were purified by PCR purification(Qiagen®) and 200 ng was denatured and re-annealed in a thermocycler anddigested with T7 Endonuclease I (New England Biolabs®) according tomanufacturer's protocol. Digested DNA were electrophoresed in a 1%agarose gel and viewed on a BioRad® ChemiDoc® imager. Band intensitieswere analyzed using the Image Lab Software (Bio-Rad®) and allelemodification frequencies (INDEL) were calculated with the formula:100×(1−(1−fraction cleaved){circumflex over ( )}0.5). For analyzingallele modification frequencies using TIDE (Tracking of In/dels byDecomposition), the purified PCR products were Sanger-sequenced (Eton™)using both PCR primers and each sequence chromatogram was analyzed withthe online TIDE software (Deskgen). Analyses were performed using areference sequence from a mock-transfected (Cas9 protein only) sample.Parameters were set to the default maximum indel size of 10 nucleotidesand the decomposition window to cover the largest possible window withhigh quality traces. All TIDE analyses below the detection sensitivityof 3.5% were set to 0%.

To determine the extent genomic deletion with dual ms-sgRNAs, endpointPCR was performed with primers flanking CRISPR cut sites that amplify an804 bp region. PCR products were electrophoresed in a 1% agarose gel andviewed on a BioRad® ChemiDoc® imager to observe the intact parental bandand the expected smaller (400-600 bp depending on ms-sgRNA combination)deletion product. Band intensities were analyzed using the Image LabSoftware (Bio-Rad®) and percent deletions were calculated with theformula: 100× fraction cleaved). Gel bands were extracted with a gelextraction kit (Qiagen®) and further purified by PCR purification(Qiagen®) for Sanger sequencing (Eton Bioscience™)

Flow Cytometry and FACS Analysis

Raji-fluc-GFP cells nucleofected with RNP as described above weremaintained in cell culture for 48 hrs. Live cells were stained withPE-conjugated CD19 antibody (IM1285U; Beckman Coulter®) and analyzedsorted on a BD FACS Aria™ by expression of CD19. CD34+ HSCs were stainedfor CD33 using an anti-CD33 antibody (P67.7) and analyzed by flowcytometry on the Attune® NxT flow cytometer (Life Technologies®).

CAR-T Cell Cytotoxicity Assays

CD19-directed CAR-T cells (CART19) were generated by transduction ofCART19-expressing lentivirus into CD4+ and CD8+ T cells from healthyhuman donors. CART19 construct contains a CD19-recognizing domain(single chain variable fragment derived from FMC63 monoclonal antibody),a costimulatory domain derived from CD28, and the CD3 zeta domain. Thecytotoxicity of CART19 was assessed by flow cytometry-based assay.Raji-fluc-GFP cells stained with CellTrace™ Violet dye served as targetcells. T cells not transduced with CART19 construct were used as anegative control for the cytotoxicity assay. The effector (E) and tumortarget (T) cells were co-cultured at the indicated E/T ratios (10:1,3:1, 0:1), with 1×10⁴ target cells in a total volume of 200 μl per wellin CTS™ OpTmizer™-based serum free medium. After 20 hours of incubation,cells were stained for Propidium Iodide and analyzed by Attune® NxT flowcytometer (Life Technologies®). Live target cells were gated asPropidium Iodide-negative and CellTrace™ Violet-positive. Cytotoxicitywas calculated as (1-(Live target cell fraction in CART19 group)/(Livetarget cell fraction in negative control group))×100%.

In Vivo Engraftment Experiments

For CD19 in vivo engraftment experiments, cells are engrafted into NODscid gamma mice (NSG™ mice; The Jackson Laboratory). For CD33 in vivoengraftment experiments, cells are engrafted into NSG™-SGM3 mice (TheJackson Laboratory).

In Vitro CFU Assay

1500 sorted CD34+ HSPCs were plated in 1.5 ml of methylcellulose(MethoCult™ H4034 Optimum, Stem Cell Technologies™) on a 35 mm cellculture dish and cultured for two weeks at 37° C. in a 5% CO2 incubator.Colonies were then counted and scored based on morphological appearance.

Results

(i) Selection of gRNAs

Exon 2 of CD19 was targeted for CRISPR/Cas9-mediated genomic deletion asexemplified in FIG. 4. A pair of sgRNAs, one sgRNA targeting intron 1and one sgRNA targeting intron 2, leads to simultaneous generation ofDNA double stranded breaks (DSBs) by Cas9 and excision of the regionincluding complete loss of exon 2 of CD19. The ends distal to the cutsite are repaired through ligation of introns 1 and 2 via non-homologousend joining (NHEJ). Transcription of the modified CD19 gene results inexpression of a CD19 variant lacking exon 2 (“CD19exon2 deletion”) viaexon 2 skipping during RNA splicing.

A panel of sgRNAs targeting introns 1 and 2 was designed by manualinspection for the SpCas9 PAM (5′-NGG-3′) with close proximity to CD19exon 2 and prioritized according to predicted specificity by maximizingon-target and minimizing potential off-target sites in the human genomewith an online search algorithm (Benchling, Doench et al (2016); Hsu etal (2013)) (Table 3). Exon 4 of CD19 may also be targeted forCRISPR/Cas9-mediated genomic deletion, for example using sgRNA-23 andsgRNA-24 (Table 3). For each of the example CD19 sgRNAs, the sequencetargets CD19 and the Cas type is SpCas9.

TABLE 3 CD19 sgRNA panel On Target Off Target (Doench et (Hsu et al NamesgRNA Sequence Location Strand PAM al 2016)¹ 2013)¹ CD19_sgRNAGAGGCTGGAAACTTGAGTTG Intron 1 1 TGG 57 67 −1 (SEQ ID NO: 14) CD19_sgRNAGAGGGTAAGTTACTCAGCCA Intron 1 −1 AGG 68 60 −3 (SEQ ID NO: 15) CD19_sgRNAAAATTCAGGAAAGGGTTGGA Intron 1 1 AGG 53 62 −4 (SEQ ID NO: 16) CD19_sgRNAAAGGGTTGGAAGGACTCTGC Intron 1 1 CGG 60 64 −5 (SEQ ID NO: 17) CD19_sgRNAAGCAGAGGACTCCAAAAGCT Intron 1 −1 GGG 62 59 −6 (SEQ ID NO: 18) CD19_sgRNACACACCAGGTTATAGAGCAG Intron 1 −1 AGG 63 67 −7 (SEQ ID NO: 19) CD19_sgRNACTGCTCTATAACCTGGTGTG Intron 1 1 AGG 71 63 −8 (SEQ ID NO: 20) CD19_sgRNAACCTGGTGTGAGGAGTCGGG Intron 1 1 GGG 58 69 −9 (SEQ ID NO: 21) CD19_sgRNACACAGCGTTATCTCCCTCTG Exon 2 −1 TGG 68 69 −10 (SEQ ID NO: 22) CD19_sgRNACGGACCTCTTCTGTCCATGG Intron 2 −1 TGG 65 65 −13 (SEQ ID NO: 23)CD19_sgRNA CCATGGACAGAAGAGGTCCG Intron 2 1 CGG 72 65 −14 (SEQ ID NO: 24)CD19_sgRNA GGGCGAAACTCGGAGCTAGG Intron 2 1 TGG 80 65 −15 (SEQ ID NO: 25)CD19_sgRNA GCTAGGTGGGCAGACTCCTG Intron 2 1 GGG 59 60 −16 (SEQ ID NO: 26)CD19_sgRNA GGAACCTCTAGTGGTGAAGG Exon 1 TGG −18 (SEQ ID NO: 69)CD19_sgRNA CACAGCGTTATCTCCCTCTG Exon 2 GGT −19 (SEQ ID NO: 70)CD19_sgRNA GGACAGGGAGAGATAAGACA Intron 4 AGG −23 (SEQ ID NO: 71)CD19_sgRNA AGGTAGAGTTTCTCTCAACT Intron 4 GGG −24 (SEQ ID NO: 72) ¹On andOff-target predictions based on the indicated published algorithms.Score is out of 100 and is a prediction of success.

For gene editing, the sgRNAs were modified as described in the Materialsand Methods. The modified sgRNAs are denoted with “ms” prefix. The CD19sgRNAs targeting either intron 1 or 2 were screened in K562 cells, ahuman leukemic cell line and analyzed by T7E1 assay and TIDE analysis(FIG. 5). Of the 12 ms-sgRNAs assessed, ms-sgRNAs 1, 3-9 target intron1, ms-sgRNA 10 targets exon 2, and ms-sgRNA 14-16 target intron 2. Thepercent INDEL for ms-sgRNA-1 was not calculated for this sample becausethe size-change between edited and unedited bands could not beaccurately distinguished using the current set of PCR primers.

Pairs of ms-sgRNAs were used to delete exon 2 of CD19 in K562 cells, anda PCR-based assay was used to detect CRISPR/Cas9-mediated genomicdeletion of CD19 exon 2 (FIG. 6). The combined activity of ms-sgRNAstargeting intron 1 (ms-sgRNAs 3, 4, 5, 6, 9) were screened incombination with ms-sgRNAs targeting intron 2 (ms-sgRNAs 14, 15, 16) togenerate genomic deletions. PCR across the genomic deletion region showsthe smaller deletion PCR product (400-560 bp) compared to the largerparental band (801 bp). The editing efficiency was quantified as percentdeletion by end-point PCR (FIG. 6, panel C).

(ii) In Vitro Characterization of CD34⁺ Cells Expressing Exon 2-DeletedCD19

The CD19 sgRNAs targeting either intron 1 or 2 were screened in CD34⁺HSCs (FIGS. 7 and 9). Pairs of ms-gRNAs were used to delete exon 2 ofCD19 in CD34⁺ HSCs. The combined activity of ms-sgRNAs targeting intron1 (ms-sgRNAs 4, 6, 9) were screened in combination with ms-sgRNAstargeting intron 2 (ms-gRNAs 14, 15, 16) to generate genomic deletions(FIGS. 8A and 8B). PCR across the genomic deletion region shows thesmaller deletion PCR product compared to the larger parental band. Theediting efficiency was quantified a percent deletion by end-point PCR.

Additional pairs of ms-gRNAs were used to delete exon 2 of CD19 in CD34⁺HSCs. The combined us of ms-sgRNAs targeting intron 1 (ms-sgRNAs 1, 6,7) in combination with ms-sgRNAs targeting intron 2 (ms-gRNAs 14, 15,16) were found to efficiently generate genomic deletions of exon 2 (FIG.10A and FIG. 10B).

PCR across the genomic deletion region shows the smaller deletion PCRproduct compared to the larger parental band. The results show highefficiency generation of HSCs expression CD19ex2 upon CRISPR editingusing the gRNA6/gRNA14 pair. FIG. 10C. In vitro CFU assay shows thatCD19ex2 HSCs did not affect in vitro differentiation. FIG. 10D. For theCFU assay, 1500 sorted CD34+ HSPCs were plated in 1.5 ml ofmethylcellulose (MethoCult™ H4034 Optimum, Stem Cell Technologies®) on a35 mm cell culture dish and cultured for two weeks at 37° C. in a 5% CO2incubator. Colonies were then counted and scored based on morphologicalappearance

(iii) Characterization of Raji B-Cell Lymphoma Cells Expressing Exon2-Deleted CD19

The CD19 sgRNAs targeting either intron 1 or 2 were also screened inRaji B-cell lymphoma cells HSCs. Pairs of ms-gRNAs were used to deleteexon 2 of CD19 (CD19ex2) in Raji B-cell lymphoma cells. The combinedactivity of ms-sgRNAs targeting intron 1 (ms-sgRNA 6) were used incombination with ms-sgRNAs targeting intron 2 (ms-gRNA 14) to generategenomic deletions. FIG. 11A. PCR across the genomic deletion regionshows the smaller deletion PCR product compared to the larger parentalband. The ex2 small deletion band represents a product resulting from analternative DNA repair event after Cas9 cleavage. The ex2 small deletionsequence encodes the same CD19 mutant with deletion of the fragmentencoded by exon 2. The editing efficiency was quantified a percentdeletion by end-point PCR. FIG. 11B.

Protein analysis of clonal lymphoma cells expression exon 2-deleted CD19(CD19ex2) was analyzed by both Western Blot using an antibody thatrecognizes the C-terminus of CD19 and Flow Cytometry using the FMC63anti-CD19 antibody (recognizing the CD19 region encoded by exon 2) or apolyclonal anti-CD19 antibody. Expression levels of exon 2-deleted CD19(CD19ex2) in clonal lymphoma cells was analyzed by both Western Blotusing an antibody that recognizes the C-terminus of CD19 (#3574 Rabbitanti-human CD19 Pab, Cell Signaling Technology®) of whole cell lysatesor flow cytometry using the FMC63 anti-CD19 antibody (FMC63 R-PE Mouseanti-human CD19 Mab, EMD Millipore®) that recognizes the CD19 regionencoded by exon 2. A polyclonal anti-CD19 antibody (#3574 Rabbitanti-human CD19 Pab, Cell Signaling Technology®) was used to identifyintracellular CD19 by intracellular staining and flow cytometry.

Expression of exon 2-deleted CD19 was observed in Raji B-cells with oneor both chromosomes edited as detected by Western Blot. FIG. 11C.Similarly, surface expression and intracellular presence of exon2-deleted CD19 were also detected in Raji B-cells by Flow Cytometry.FIG. 11D.

Further, growth and viability of Raji B-cells expressing CD19exon2 wereanalyzed. Human lymphoma cell line, Raji, was obtained from the AmericanType Culture Collection® (ATCC®). Raji cells were cultured in RPMI-1640media (ATCC®) supplemented with 10% heat-inactivated HyClone™ FetalBovine Serum (GE Healthcare®). Cell viability and live cell number weremeasured by an automated cell counter, Cellmoter (Nexcelom™). As shownin FIG. 11E, cells expressing CD19exon2 did not show defects inproliferation (left panel) and viability (right panel).

(iv) Engraftment of CD19ex2 Hematopoietic Stem Cells (HSCs) in aHumanized Mouse Model

An exemplary flowchart for generating HSCs expression CD19ex2 andcharacterization of the in vivo differentiation of the HSCs in ahumanized mouse model (e.g., NSG mice) is provided in FIG. 12A. In someexamples, CAR-T treatment can be performed at 14w after HSPCengraftment. The NSG mice can be assigned the four groups shown in Table4 below:

TABLE 4 In vivo characterization groups Group Condition Value EndpointExpectation 1 RNP Control + Mock EP control Functional B Cell CAR-T 2CD19ex2 + Mock Target Functional CAR-T FL/CD19ex2 B Cell 3 RNP control +CAR-T selectivity B-Cell Loss CART19 4 CD19ex2 + CART19 CAR-Tselectivity Retained B-Cell

CD34⁺ HSC cells expressing CD19ex2 were produced using the combinationof ms-sgRNAs targeting intron 1 (ms-sgRNA 6) and ms-sgRNAs targetingintron 2 (ms-gRNA 14) to generate genomic deletions as described above.See also the Materials and Methods. Samples were split into twofractions: 2% of cells were characterized in vitro and the remainingfraction is engraftment into 6-8 week old NOD scid gamma mice(NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wj1)/SzJ (NSG mice), a humanized mousemodel. The Jackson Laboratory. The in vitro fraction was characterizedby colony forming unit (CFU) assay and genotyping as described above.

The in vivo fraction was administered to irradiated NSG™ mice. Bloodsamples can be obtained from the mice at various time points (e.g., 4weeks, 8 weeks, 12 weeks) and analyzed by genotyping and to assess thepercentage human CD45+ cells. At 16 weeks, the mice can be sacrificedand peripheral blood, bone marrow, and spleens were harvested foranalysis. The primary endpoint is percent engraftment, which can beassessed by genotyping and flow cytometric analysis (e.g., mouse vshuman CD45, CD20/CD19, CD19 deficient in exon 2, Cd34, CD33, CD3). Asecondary endpoint can be expression of CD19 that is deficient in exon 2by Western blotting and/or qRT-PCR.

Percentage of hCD45+ cells in peripheral blood collected from theHSC-treated mice was determined by flow cytometry and the results areprovided in FIG. 12B. The edited HSCs expressing CD19ex2 showedefficient engraftment in the NSG mice at week 4, 9 and 12 at levelssubstantially the same as the control HSC cells.

The levels of CD45+/CD20+ cells, representing differentiated B cells, inperipheral blood collected from the NSG™ mice were measured at week 9after infusion of RNP HSCs or CD19ex2 HSCs. As shown in FIG. 12C, theedited cells (CD19ex2 HSCs) showed a significantly higher level of Bcell differentiation than the unedited cells (RNP HSCs) at 9 and 12weeks.

(v) In vivo Raji Tumor Model

An in vivo Raji tumor model can be used to assay the efficacy of any ofthe treatment methods described herein. Raji-fluc-GFP cells expressingendogenous CD19 deficient in exon 2 (CD19exon2 delete) were generated exvivo as described in the Materials and Methods. Following enrichment ofedited cells, samples are split into two fractions: one fraction ischaracterized in vitro and the remaining fraction is xenografted into6-8 week old NSG™ mice (FIG. 13).

The in vitro fraction is characterized by cytotoxicity and molecularassays as described in the Materials and Methods. The in vivo fractionis assessed for efficacy and selectivity of CART19 in Burkett Lymphomamouse model and assayed by the indicated assays and as described inMaterials and Methods. The groups of mice are shown in Table 5. Briefly,one week following injection of the Raji-fluc-GFP cells expressingendogenous CD19 deficient in exon 2, the mice are infused CART19 cells.The mice are assessed at various time points (e.g., 6 days, 12 days, 18days, 35 days) by in vivo imaging system (IVIS) to determine theabundance of Raji cells (CD19/CD19ex2). Blood samples are also obtainedfrom the mice to quantify the number of CART19 cells.

TABLE 5 In vivo characterization groups Group Condition CART19 # Mice 1Untreated control −  4 2 Untreated control + 10 3 Raji Fluc GFP; CD19+/+− 10 4 Raji Fluc GFP; CD19+/+ + 10 5 Raji Fluc GFP; CD19exon2DEL − 10 6Raji Fluc GFP; CD19exon2DEL + 10

The primary endpoint of treatment efficacy is assessed, for example, bysurvival, tumor burden volume, and tumor burden by IVIS imaging. Theprimary endpoint of treatment selectivity is assessed, for example, bydetermining persistence of Raji-GFP cells. Secondary endpoints forCART19 therapy include pharmacokinetics and tumor infiltration, andsecondary endpoints for CD19 include expression of CD19 that isdeficient in exon 2.

It is expected that Raji cells expressing exon 2 of CD19 will be killedby the CART19 cells, whereas the Raji cells that have been manipulatedto delete exon 2 of CD19 will survive and evade CART killing.

(vi) Generation of Raji-Fluc-GFP Cells Lines Deficient in CD19 Exon 2

Raji-fluc-GFP cell lines were transfected with pairs of ms-sgRNAs andassayed for CD19 expression by fluorescence-activated cell sorting(FACS). Cells were gated into three populations based on relative CD19expression: “hi” (high), “int” (intermediate), and “lo” (low) (FIG.14A). Parental Raji cells and Raj-fluc-GFP nucleofected with Cas9 onlywere included as controls. The percentage of live cells in eachcondition was quantified (FIG. 14B). PCR was also performed across thegenomic deletion region of cells in each condition showing the smallerdeletion PCR product compared to the larger parental band (FIG. 14C).The percentage CD19 exon 2 in the bulk population was also assayed byend-point PCR in each condition (FIG. 14D), indicating there was ahigher percentage of cells with the CD19 exon 2 deletion in the CD19“int” and CD19 “lo” cell populations.

(vii) CART Cytotoxicity

CD19-directed CAR-T cells (CART19) were generated as described in theMaterials and Methods and incubated with Raji-fluc-GFP cells. Following20 hours of incubation, cytotoxicity was assessed by flow cytometry.FIG. 15A and FIG. 15B shows there was reduced specific lysis of CD19“low” Raji cells as compared to CD19 “hi” populations.

As shown in FIGS. 14A-14D, the Raji “hi” population is genotypicallymixed population of cells. Single cells may be enriched to analyzeclonal populations as well as unedited parental populations. The controlCD19-hi population is a mixed genotype (20-40% CD19exon2 delete), andenhanced killing is expected with wild-type control populations.

(viii) In Vivo Efficacy and Selectivity

FIG. 16 outlines a comprehensive in vivo model assessing efficacy andselectivity of CART therapy paired with edited HSCs. Briefly, HSCsdeficient in exon 2 of CD19 (CD19ex2delete) are prepared. Groups of miceare administered either control (unedited) HSCs or HSCs deficient inexon 2 of CD19. After four weeks, the mice are administered RajiBurkitt's lymphoma cells, followed by CART19 cells one week later. Themice are assessed weekly by IVIS® imaging, and blood samples areobtained every four weeks. After 12 weeks, the mice are sacrificed andperipheral blood, bone marrow, and spleens are harvested for analysis.

Example 2: Deletion Exon 2 of CD33 Via CRISPR/Cas9-Mediated Gene Editingand Characterization of Cells Expressing Exon 2-Deleted CD33

This Example reports genetic engineering of CD33 genes via CRISPR/Cas9in cells to produce edited cells expressing mutated CD33, in which thefragment encoded by exon 2 is deleted (CD33ex2), and in vitro and invivo characterization of such edited cells using assays described inExample 1 above or known in the art. See also above disclosures forexemplary exon-2 deleted CD33 gene products.

(i) Selection of gRNAs

As shown in FIG. 17, the Cas9 nuclease is targeted to introns 1 and 2 ofCD33 by two sgRNAs. Simultaneous generation of DNA double strandedbreaks (DSBs) by Cas9 leads to excision of the region including completeloss of exon 2. The ends distal to the cut site are repaired throughligation of introns 1 and 2 via non-homologous end joining (NHEJ) withthe repaired junction indicated by the triangle. Transcription of themodified genome results in expression of CD33m isoform.

A panel of ms-sgRNAs was designed by manual inspection for the SpCas9PAM (5′-NGG-3′) with close proximity to CD33 exon 2 and prioritizedaccording to predicted specificity by minimizing potential off-targetsites in the human genome with an online search algorithm (Benchling,Doench et al (2016); Hsu et al (2013)) (Table 6). A subset of ms-sgRNAstargeting either intron 1 or 2 was then selected based on in vitro geneediting efficiency. Each of the sgRNAs target human CD33 and use Cas9type SpCas9.

TABLE 6 CD33 sgRNA panel On Target Off Target (Doench et al (Hsu et alName sgRNA Sequence PAM Location 2016)¹ 2013)¹ CD33_sgRNAGCTGTGGGGAGAGGGGTTGT CGG Intron 1 39 29 −1 (SEQ ID NO: 27) CD33_sgRNACTGTGGGGAGAGGGGTTGTC GGG Intron 1 46 35 −2 (SEQ ID NO: 28) CD33_sgRNATGGGGAAACGAGGGTCAGCT CGG Intron 1 60 29 −3 (SEQ ID NO: 29) CD33_sgRNAGGGCCCCTGTGGGGAAACGA GGG Intron 1 65 40 −4 (SEQ ID NO: 30) CD33_sgRNAAGGGCCCCTGTGGGGAAACG AGG Intron 1 50 36 −5 (SEQ ID NO: 31) CD33_sgRNAGCTGACCCTCGTTTCCCCAC AGG Intron 1 47 31 −6 (SEQ ID NO: 32) CD33_sgRNACTGACCCTCGTTTCCCCACA GGG Intron 1 52 27 −7 (SEQ ID NO: 33) CD33_sgRNATGACCCTCGTTTCCCCACAG GGG Intron 1 71 29 −8 (SEQ ID NO: 34) CD33_sgRNACCATAGCCAGGGCCCCTGTG GGG Intron 1 61 24 −9 (SEQ ID NO: 35) CD33_sgRNAGCATGTGACAGGTGAGGCAC AGG Intron 2 56 36 −10 (SEQ ID NO: 36) CD33_sgRNATGAGGCACAGGCTTCAGAAG TGG Intron 2 55 32 −11 (SEQ ID NO: 37) CD33_sgRNAAGGCTTCAGAAGTGGCCGCA AGG Intron 2 54 39 −12 (SEQ ID NO: 38) CD33_sgRNAGGCTTCAGAAGTGGCCGCAA GGG Intron 2 58 44 −13 (SEQ ID NO: 39) CD33_sgRNAGTACCCATGAACTTCCCTTG CGG Intron 2 75 40 −14 (SEQ ID NO: 40) CD33_sgRNAGTGGCCGCAAGGGAAGTTCA TGG Intron 2 63 42 −15 (SEQ ID NO: 41) CD33_sgRNATGGCCGCAAGGGAAGTTCAT GGG Intron 2 53 43 −16 (SEQ ID NO: 42) CD33_sgRNAGGAAGTTCATGGGTACTGCA GGG Intron 2 66 42 −17 (SEQ ID NO: 43) CD33_sgRNATTCATGGGTACTGCAGGGCA GGG Intron 2 59 32 −18 (SEQ ID NO: 44) CD33_sgRNACTAAACCCCTCCCAGTACCA GGG Intron 2 61 40 −19 (SEQ ID NO: 45) CD33_sgRNACACTCACCTGCCCACAGCAG GGG Intron 1 56 23 −20 (SEQ ID NO: 46) CD33_sgRNACCCTGCTGTGGGCAGGTGAG TGG Intron 1 44 20 −21 (SEQ ID NO: 47) CD33_sgRNATGGGCAGGTGAGTGGCTGTG GGG Intron 1 61 26 −22 (SEQ ID NO: 48) CD33_sgRNAGGTGAGTGGCTGTGGGGAGA GGG Intron 1 42 24 −23 (SEQ ID NO: 49) CD33_sgRNAGTGAGTGGCTGTGGGGAGAG GGG Intron 1 49 20 −24 (SEQ ID NO: 50) ¹On andOff-target predictions based on the indicated published algorithms.Score is out of 100 and is a prediction of success.

The CD33 ms-sgRNAs targeting introns 1 or 2 were screened in primaryCD34⁺ HSCs by TIDE assay (FIGS. 17A and 17B).

Pairs of ms-gRNAs were used tested in CD34+ HSCs (FIGS. 18B and 18C;FIG. 27). Efficient deletion of exons 2 and 3, leading to knock-out ofCD33, was observed using control sgRNAs targeting exons 2 and 3 (Sg and811, respectively). A reduction in CD33 containing exon 2 was observedwith pairs of sgRNAs targeting introns 1 and 2 (e.g., sgRNAs 17 and 23;sgRNAs 18 and 24).

FIG. 19A shows results from flow cytometry showing CD34+ HSCs, eitherunedited (left panel, mock) or edited with guide RNAs for KO (producinga full CD33 knockout (middle panel) or edited with guide RNAs 18 and 24resulting in the expression of mutated CD33 with exon 2-encoded fragmentdeleted (CD33ex2, right panel). FIG. 19B shows the editing efficiency asdetermined from the flow cytometric analysis shown in FIG. 19A. Thepercentage of CD34+ CD33ex2 events was determined using deletion PCRcontaining primers flanking exon 2 (shown in black).

(ii) Genotyping and In Vitro Differentiation of Dual gRNA-TargetedCD33ex2 Cells

CFU assays of cells edited by the pair of gRNA18 and gRNA24, as well asCD33-KO CD34⁺ HSC cells show that both type of cells retaindifferentiation potential in vitro. FIG. 20A. PCR genotyping of a numberof differentiated myeloid cell clones expressing the CD33ex2 mutant showthat the pair of gRNA18 and gRNA24 resulted in a mixture of exon 2deleted CD33 alleles and CD33 knocked-out alleles. FIG. 19B and FIG.20B. The genotypes and frequencies are provided in Table 7 below.

TABLE 7 Genotype and Frequency of Differentiated CD33ex2 Myeloid CellsGenotype Frequency KO/KO 44 of 87 ex2/ex2 16 of 87 ex2/KO 27 of 87

(iii) Generation and Characterization of CD33ex2 in AML Cell Lines

AML cell lines THP1 (monocytic leukemia) and HL-60 (promyeloblastleukemia) were genetically edited via CRISPR/Cas9 using the pair ofgRNA18 and gRNA24 to produce cells expressing CD33ex2, which were sortedby flow cytometry. Single cells were expanded to establish clones thatexpress CD33ex2, which were subject to gemtuzumab ozogamicin treatmentas disclosed below. Genomic PCR showed that the CD33 exon 2 was deletedeither in one CD33 allele or in both alleles. FIG. 21A. FIG. 21Billustrates the PCR primer pairs for detecting total CD33, includingboth full length (M) and exon 2 deletion (m) and the PCR primer pairsfor detecting only CD33m. One clone shows partial deletion in the exon 1region. The levels of full-length CD33 transcripts and CD33ex2transcripts were analysed by an RNA analysis and the results are shownin FIG. 21C. Deeper genomic characterization via PCR and sequencinganalysis show reproducible repair ligation of intron 1 and intron 2.Depending on the type of IDEL the repair ligation results in eithertranscripts resulting in CD33ex2 or full CD33 KO. For example, in somecases the INDELS extend into exon 1, resulting in a CD33 KO.

(iv) CD33ex2 Cells are Less Susceptible to Gemtuzumab Ozogamicin (GO)Cytotoxicity

The AML cell clones expressing CD33ex2 were treated with gemtuzumabozogamicin and the viability of the treated cells was determined. HumanAML cell lines, THP-1 and HL-60, were obtained from the American TypeCulture Collection® (ATCC®). THP-1 cells were cultured in RPMI-1640media (ATCC) supplemented with 10% heat-inactivated HyClone™ FetalBovine Serum (GE Healthcare®) and 0.05 mM 2-mercaptoethanol(Sigma-Aldrich®). HL-60 cells were cultured in Iscove's ModifiedDulbecco's Medium (IMDM, Gibco™) supplemented with 20% heat-inactivatedHyClone™ Fetal Bovine Serum (GE Healthcare®). For in vitro cytotoxicityassays with AML cell lines, THP-1 and HL-60 cells were incubated with GOin their complete culture media for 72 hours. Increasing concentrationsof GO, from 0.1 ng/mL to 10 μg/mL, were used. Cells were plated at 2×104per well in 96 well plates. Viable cell number was measured withCelltiter Glo (Promega) according to manufacturer's instructions, andluminescence was determined using a GloMax multiplate reader (Promega).IC50 was defined as the concentration of compound needed to yield a 50%reduction in viable cell number compared with no GO treated cells(control=100%). To measure cell death, cells were stained with Annexin V(Invitrogen®)) and DAPI (Biolegend) for 15 minutes in 1× Annexin BindingBuffer (Invitrogen®) at room temperature. Data was acquired on AttuneNxT flow cytometer (ThermoFisher Scientific®), and analyzed with FlowJo®software (TreeStar).

As shown in FIG. 22A, AML cell lines THP-1 and HL-60 are moresusceptible to GO cytotoxicity (IC₅₀ 16.3 ng/ml and 35.5 ng/ml,respectively) as compared with Jurkat cells (IC₅₀ 902.7 ng/ml). EditedAML cells expressing CD33ex2 showed lower susceptibility to GOcytotoxicity as compared with the unedited parent cells. FIG. 22B.

Similar results were observed in edited HSPC cells expressing CD33ex2.In one study, the IC50 value of wild-type HSPC was found to be around93.4 ng/ml, and the IC₅₀ values of HSPC CD33KO and HSPC CD33ex2 werefound to be around 248.1 ng/ml and 246.4 ng/ml, respectively.

Wild-type HSC cells (CD33+), CD33 KO HSCs and CD33ex2 HSCs were treatedwith GO and the surviving cells were cultured in a myeloid culturemedium for 30 days after withdrawal of GO treatment. The viability ofthe surviving cells was determined as described above. Surprisingly,CD33ex2 HSCs showed much higher viability post GO treatment, as comparedwith wild-type and CD33 KO HSC cells. FIG. 22C.

(v) CD33ex2 Cells are Protected from CART33-Mediated Killing

CD33-directed CAR-T cells (CART33 or CAR1) were generated bytransduction of CART33-expressing lentivirus into CD4⁺ and CD8⁺ T cellsfrom healthy human donors. The CART33 construct contains a single chainvariable fragment (scFv) that recognizes the fragment of CD33 encoded byexon 2, a hinge domain of CD8α, a transmembrane domain of CD8, aco-stimulatory domain of 4-1BB, and a cytoplasmic signaling domain ofCD3ζ.

The cytotoxicity of CART33 was assessed by flow cytometry-based assay.Raji (CD33⁻) cells were used as a negative control for the cytotoxicityassay. The effector (E) and tumor target (T) cells were co-cultured atthe indicated E/T ratios (5:1 and 1:1), with 1×10⁴ target cells in atotal volume of 200 μl per well in CTS™ OpTmizer™-based serum freemedium. After 20 hours of incubation, cells were stained for PropidiumIodide and analyzed by Attune® NxT flow cytometer (Life Technologies®).Live target cells were gated as Propidium Iodide-negative and CellTrace™Violet-positive. Cytotoxicity was calculated as (1-(Live target cellfraction in CART19 group)/(Live target cell fraction in negative controlgroup))×100%.

A high level of cell lysis was observed in CD33⁺HL-60 cells (FIG. 23A)and expression of CD33ex2 mutant in HL-60 cells significantly reducedthe level of cell lysis (FIG. 23B), similar to HL-60 cells having CD33knocked-out (FIG. 23C). The comparison results are provided in FIG. 23D.

Example 3: Efficient Multiplex Genomic Editing

Efficient double genomic editing of CD19 and CD33 genes in HSC cellswere performed in either NALM-6 cells or in HSCs following conventionalmethods or those described herein. Table 8 below provides the gRNAstargeting exon 2 of CD19 and exon 3 of CD33.

TABLE 8 Guide RNAs for Double Editing of CD19 and CD33 Gene gRNA NamegRNA Sequence PAM Location CD19 CD19_gRNA- CACAGCGTTATCTCCCTCTG GGTExon 2 19 (SEQ ID NO: 66) CD33 CD33_gRNA- CCCCAGGACTACTCACTCCT CGGExon 3 37 (SEQ ID NO: 67)

Genomic DNA was isolated from bulk-edited cells and TIDE assays wereperformed to examine genomic editing in NALM-6, HL-60 cells and HSCs.Results are depicted in FIGS. 24A-24D. The results obtained from thisstudy show that ˜70% of the HSCs include mutations in both loci of theCD19 gene and ˜80% of the HSCs include mutations in both loci of theCD33 gene, indicating that at least 50% of the double-edited cells haveboth edited CD19 gene and edited CD33 gene on at least one chromosome.Similar levels of edited cells were observed in HL-60 cells and Nalm-6cells.

Example 4: Effect of Editing Multiple Loci on Viability

The effect of genomic editing at multiple loci on cell viability wasassessed NALM-6, HL-60 cells and HSCs. Two days before nucleofection,HSPCs were thawed or Nalm-6 or HL-60 cells were passaged. At 24 and 48hours (day of nucleofection), cells were counted and cell viability wasassessed. Nucleofection with CD33_gRNA-37 RNP and CD19_gRNA-19 complexesand was performed according to Materials and Methods described herein.Cells were counted and cell viability was assessed at the indicatedtimepoints. Results are depicted in FIGS. 25A-25C and indicate that dualCas9/gRNAs delivery does not impair viability in cell lines. Noadditional toxicity over single guide RNA was observed in HSCs.

Example 5: Performance of Individual Guides

The efficiency of genomic editing for the individual guide RNAs CD33sgRNA-18 and CD33 sgRNA-24 was assessed in HL-60 cells. Genomic DNA wasisolated from bulk-edited cells and TIDE assays were performed toexamine genomic editing. Results are depicted in Table 9.

TABLE 9 Percentage of INDELs resulting from gRNA 18 and gRNA 24individually CD33 Guide Indel in HL60 cells 3′-sg 18 94% 5′-sg-24 46%

Example 6: Treatment of Hematologic Disease

An example treatment regimen using the methods, cells, and agentsdescribed herein for acute myeloid leukemia is provided below.

1) Identify a patient with AML that is a candidate for receiving ahematopoietic cell transplant (HCT);

2) Identify a HCT donor with matched HLA haplotypes, using standardmethods and techniques;

3) Extract the bone marrow from the donor;

4) Genetically manipulate the donor bone marrow cells ex vivo. Briefly,introduce a targeted modification (deletion, substitution) of an epitopeof the lineage-specific cell-surface antigen n. In general, the epitopeshould generally be at least 3 amino acids (e.g., about 6-10 aminoacids). Genetic modification of this epitope of the targetedlineage-specific cell-surface antigen on the donor bone marrow cellsshould not substantially impact the function of the protein, and as aconsequence, should not substantially impact the function of the bonemarrow cells, including their ability to successfully engraft in thepatient and mediate graft-vs-tumor (GVT) effects;

Optional Steps 5-7:

In some embodiments, Steps 5-7 provided below may be performed (once ormultiple times) in an exemplary treatment method as described herein:

5) Pre-condition the AML patient using standard techniques, such asinfusion of chemotherapy agents (e.g., etoposide, cyclophosphamide)and/or irradiation;

6) Administer the engineered donor bone marrow to the AML patient,allowing for successful engraftment;

7) Follow up with a cytotoxic agent, such as immune cells expressing achimeric receptor (e.g., CAR T cell) or antibody-drug conjugate, whereinthe epitope to which the cytotoxic agent binds is the same epitope thatwas modified and is no longer present on the donor engineered bonemarrow graft. The targeted therapy should thus specifically target theepitope of the lineage-specific cell-surface antigen, withoutsimultaneously eliminating the bone marrow graft, in which the epitopeis not present;

Optional Steps 8-10:

In some embodiments, Steps 8-10 may be performed (once or multipletimes) in an exemplary treatment method as described herein:

8) Administer a cytotoxic agent, such as immune cells expressing achimeric receptor (e.g., CAR T cell) or antibody-drug conjugate thattargets an epitope of a lineage-specific cell-surface antigen. Thistargeted therapy would be expected to eliminate both cancerous cells aswell as the patient's non-cancerous cells;

9) Pre-condition the AML patient using standard techniques, such asinfusion of chemotherapy agents;

10) Administer the engineered donor bone marrow to the AML patient,allowing for successful engraftment.

The steps 8-10 result in the elimination of the patient's cancerous andnormal cells expressing the targeted protein, while replenishing thenormal cell population with donor cells that are resistant to thetargeted therapy.

Example 7: Effect of Gemtuzumab Ozogamycin on Engineered HSCs

As shown in FIG. 28, frozen CD34+HSPCs derived from mobilized peripheralblood were thawed and cultured for 72 h before electroporation withribonucleoprotein comprising Cas9 and an sgRNA. Samples wereelectroporated with the following conditions:

i.) Mock (Cas9 only),

ii. KO sgRNA (CD33 sgRNA-37), or

iii. Exon2deletion (dual gRNA 18+24).

Cells were allowed to recover for 72 hours and genomic DNA was collectedand analyzed by PCR of the gRNA target region. To determine thepercentage of cells having an exon 2 knockout were assessed by TIDE, andthe percentage INDEL was determined. The fraction of the population withdeletion of exon 2 was assayed by end-point PCR.

The percentage of CD33-positive cells were assessed by flow cytometry,confirming that editing with gRNA-37 was effective in knocking out CD33.FIG. 30.

The editing events in the HSCs were found to result in a variety ofindel sequences, as exemplified in FIG. 29.

(i) Sensitivity of Cells Having CD33exon2 Deletion to GemtuzumabOzogramicin (GO)

To determine in vitro toxicity, cells were incubated with GO in theirculture media and the number of viable cells was quantified over time.As shown in FIGS. 31A and 31B, CD33 knockout cells generated with CD33sgRNA-37 and CD33ex2 del cells generated with the CD33 gRNA-18 andgRNA-24 pair were more resistant to GO treatment than cells expressingfull length CD33 (mock). 50% editing observed in CD33KO cells isconsidered sufficient protection in dividing cells. The initial declinein the viability of CD33ex2 del cells is thought to correspond tounedited cell death.

(ii) Enrichment of CD33-Modified Cells

To assay if CD33 modified cells were enriched following GO-treatment,CD34+ HSPCs were edited with 50% of standard Cas9/gRNA ratios. The bulkpopulation of cells were analyzed prior to and after GO treatment. Asshown in FIG. 31C, prior to GO treatment, 51% of gRNA-37 modified cells(KO) as assayed by TIDE and 15% of gRNA 19+24 (ex2deletion) as assayedby deletion PCR. Following GO-treatment, CD33 modified cells wereenriched at least 1.5× so that the percentage of KO cells increased to80% and exon2delete to 50%. This data indicated that there was anenrichment of CD33 modified cells following GO-treatment.

(iii) In Vitro Differentiation of CD34+ HSPCs

Cell populations were assessed for myeloid differentiation prior to andafter GO treatment at various days post differentiation. As shown inFIGS. 31D and 31E, CD33 knockout cells generated with CD33 sgRNA-37 andCD33ex2 del cells generated with the CD33 gRNA-18 and gRNA-24 pairshowed increased expression of the differentiation marker, CD14, whereascells expressing full length CD33 (mock) did not differentiate.

Example 8: Generation of Exon 2 or Exon 4 Deletion in CD19

NALM6 cell lines were transfected with indicated ms-sgRNAs to modify theCD19 locus as follows:

1) WT (wild-type, unmodified),

2) sgRNA 6+14 targeting introns 1 and 2 to generate theCD19exon2deletion protein,

3) sgRNA 7+16 targeting introns 1 and 2 to generate theCD19exon2deletion protein of CD19,

4) sgRNA 23+24 targeting introns 3 and 4 to generate theCD19exon4deletion protein and

5) sgRNA 18 targeting exon 1 to create CD19 knock out.

Transfected cell lines were clonally selected and assayed for CD19expression by fluorescence-activated cell sorting (FACS). NALM6 cellsnucleofected with ribonucleoprotein of Cas9 and the sgRNA(s), asdescribed herein, were maintained in cell culture for 48 hours.

Samples from the cell populations were assessed by Western blot usingtwo different antibodies—one recognizing the Ig-like C2-type domainencoded by exon 4 and the second the C-terminus of CD19. Proteinanalysis of clonal NALM6 cells expression full-length or those modifiedwith gRNA as indicated (NT=non-targeting control gRNA, WT=wild type). Toconfirm deletion of CD19 in gRNA nucleofected cells and modified CD19 inthe CD19ex2deletion and CD19ex4deletion clones, lysates were blottedusing a polyclonal anti-CD19 antibody (#3574 Rabbit anti-human CD19 Pab,Cell Signaling Technology®) that recognizes the CD19 C-terminus. Westernblot was performed on cells lysates with anti-CD19 antibody (OTI3B10,Origene®) that recognizes the epitope encoded by exon 4 to confirmdeletion of exon 4 in gRNA 23+24 nucleofected cells. Western blot withthe C-terminus antibody confirms that CD19ex2delete and CD19ex4deleteproteins are smaller than full length CD19. FIGS. 32A and 32B.

Example 9: Generation and Evaluation of Cells Edited for Two CellSurface Antigens

Results

Cell surface levels of CD33, CD123 and CLL1 (CLEC12A) were measured inunedited MOLM-13 cells and THP-1 cells (both human AML cell lines) byflow cytometry. MOLM-13 cells had high levels of CD33 and CD123, andmoderate-to-low levels of CLL1. HL-60 cells had high levels of CD33 andCLL1, and low levels of CD123 (FIG. 33).

CD33 and CD123 were mutated in MOLM-13 cells using gRNAs and Cas9 asdescribed herein, CD33 and CD123-modified cells were purified by flowcytometric sorting, and the cell surface levels of CD33 and CD123 weremeasured. CD33 and CD123 levels were high in wild-type MOLM-13 cells;editing of CD33 only resulted in low CD33 levels; editing of CD123 onlyresulted in low CD123 levels, and editing of both CD33 and CD123resulted in low levels of both CD33 and CD123 (FIG. 34). The editedcells were then tested for resistance to CART effector cells using an invitro cytotoxicity assay as described herein. All four cell types(wild-type, CD33^(−/−), CD123^(−/−), and CD33^(−/−)CD123^(−/−))experienced low levels of specific killing in mock CAR controlconditions (FIG. 35, leftmost set of bars). CD33 CAR cells effectivelykilled wild-type and CD123^(−/−) cells, while CD33^(−/−) andCD33^(−/−)CD123^(−/−) cells showed a statistically significantresistance to CD33 CAR (FIG. 35, second set of bars). CD123 CAR cellseffectively killed wild-type and CD33^(−/−) cells, while CD123^(−/−) andCD33^(−/−)CD123^(−/−) cells showed a statistically significantresistance to CD123 CAR (FIG. 35, third set of bars). A pool of CD33 CARand CD123 CAR cells effectively killed wild-type cells, CD33^(−/−)cells, and CD123^(−/−) cells, while CD33^(−/−)CD123^(−/−) cells showed astatistically significant resistance to the pool of CAR cells (FIG. 35,rightmost set of bars). This experiment demonstrates that knockout oftwo antigens (CD33 and CD123) protected the cells against CAR cellstargeting both antigens. Furthermore, the population of edited cellscontained a high enough proportion of cells that were edited at bothalleles of both antigens, and had sufficiently low cell surface levelsof cell surface antigens, that a statistically significant resistance toboth types of CAR cells was achieved.

CD33 and CLL1 were mutated in HL-60 using gRNAs and Cas9 as describedherein, CD33 and CLL1-modified cells were purified by flow cytometricsorting, and the cell surface levels of CD33 and CLL1 were measured.CD33 and CLL1 levels were high in wild-type HL-60 cells; editing of CD33only resulted in low CD33 levels; editing of CLL1 only resulted in lowCLL1 levels, and editing of both CD33 and CLL1 resulted in low levels ofboth CD33 and CLL1 (FIG. 36). The edited cells were then tested forresistance to CART effector cells using an in vitro cytotoxicity assayas described herein. All four cell types (wild-type, CD33^(−/−),CLL1^(−/−), and CD33^(−/−) CLL1^(−/−)) experienced low levels ofspecific killing in mock CAR control conditions (FIG. 37, leftmost setof bars). CD33 CAR cells effectively killed wild-type and CLL1^(−/−)cells, while CD33^(−/−) and CD33^(−/−) CLL1^(−/−) cells showed astatistically significant resistance to CD33 CAR (FIG. 37, second set ofbars). CLL1 CAR cells effectively killed wild-type and CD33^(−/−) cells,while CLL1^(−/−) and CD33^(−/−) CLL1^(−/−) cells showed a statisticallysignificant resistance to CLL1 CAR (FIG. 37, third set of bars). A poolof CD33 CAR and CLL1 CAR cells effectively killed wild-type cells,CD33^(−/−) cells, and CLL1^(−/−) cells, while CD33^(−/−) CLL1^(−/−)cells showed a statistically significant resistance to the pool of CARcells (FIG. 37, rightmost set of bars). This experiment demonstratesthat knockout of two antigens (CD33 and CLL1) protected the cellsagainst CAR cells targeting both antigens. Furthermore, the populationof edited cells contained a high enough proportion of cells that wereedited at both alleles of both antigens, and had sufficiently low cellsurface levels of cell surface antigens, that a statisticallysignificant resistance to both types of CAR cells was achieved.

The efficiency of gene editing in human CD34+ cells was quantified usingTIDE analysis as described herein. At the endogenous CD33 locus, editingefficiency of between about 70-90% was observed when CD33 was targetedalone or in combination with CD123 or CLL1 (FIG. 38, left graph). At theendogenous CD123 locus, editing efficiency of between about 60% wasobserved when CD123 was targeted alone or in combination with CD33 orCLL1 (FIG. 38, center graph). At the endogenous CLL1 locus, editingefficiency of between about 40-70% was observed when CLL1 was targetedalone or in combination with CD33 or CD123 (FIG. 38, right graph). Thisexperiment illustrates that human CD34+ cells can be edited at a highfrequency at two cell surface antigen loci.

The differentiation potential of gene-edited human CD34+ cells asmeasured by colony formation assay as described herein. Cells edited forCD33, CD123, or CLL1, individually or in all pairwise combinations,produced BFU-E colonies, showing that the cells retain significantdifferentiation potential in this assay (FIG. 39A). The edited cellsalso produced CFU-G/M/GM colonies, showing that the cells retaindifferentiation potential in this assay that is statisticallyindistinguishable from the non-edited control (FIG. 39B). The editedcells also produced detectable CFU-GEMM colonies (FIG. 39C). Takentogether, the differentiation assays indicate that human CD34+ cellsedited at two loci retain the capacity to differentiate into variety ofcell types.

Materials and Methods

AML Cell Lines

Human AML cell line HL-60 was obtained from the American Type CultureCollection (ATCC). HL-60 cells were cultured in Iscove's ModifiedDulbecco's Medium (IMDM, Gibco™) supplemented with 20% heat-inactivatedHyClone™ Fetal Bovine Serum (GE Healthcare®). Human AML cell lineMOLM-13 was obtained from AddexBio Technologies™. MOLM-13 cells werecultured in RPMI-1640 media (ATCC®) supplemented with 10%heat-inactivated HyClone™ Fetal Bovine Serum (GE Healthcare®).

Guide RNA Design

All sgRNAs were designed by manual inspection for the SpCas9 PAM(5′-NGG-3′) with close proximity to the target region and prioritizedaccording to predicted specificity by minimizing potential off-targetsites in the human genome with an online search algorithm (Benchling,Doench et al 2016, Hsu et al 2013). All designed synthetic sgRNAs werepurchased from Synthego® with chemically modified nucleotides at thethree terminal positions at both the 5′ and 3′ ends. Modifiednucleotides contained 2′-O-methyl-3′-phosphorothioate (abbreviated as“ms”) and the ms-sgRNAs were HPLC-purified. Cas9 protein was purchasedfrom Aldervon®.

Target Target gene gRNA name Sequence PAM location CD33 CD33-CCCCAGGACTACTCACTCCT CGG CD33 gRNA37 (SEQ ID NO: 134) exon 3 CD123CD123- TTTCTTGAGCTGCAGCTGGG CGG CD123 gRNA19 (SEQ ID NO: 135) exon 5CD123- AGTTCCCACATCCTGGTGCG GGG CD123 gRNA25 (SEQ ID NO: 136) exon 6CLL1 CLL1- GGTGGCTATTGTTTGCAGTG TGG CLL1 gRNA4 (SEQ ID NO: 137) exon 4AML Cell Line Electroporation

Cas9 protein and ms-sgRNA (at a 1:1 weight ratio) were mixed andincubated at room temperature for 10 minutes prior to electroporation.MOLM-13 and HL-60 cells were electroporated with the Cas9ribonucleoprotein complex (RNP) using the MaxCyte® ATx™ ElectroporatorSystem with program THP-1 and Opt-3, respectively. Cells were incubatedat 37° C. for 5-7 days until flow cytometric sorting.

Human CD34+ Cell Culture and Electroporation

Cryopreserved human CD34+ cells were purchased from Hemacare® and thawedaccording to manufacturer's instructions. Human CD34+ cells werecultured for 2 days in GMP SCGM media (CellGenix™) supplemented withhuman cytokines (Flt3, SCF, and TPO, all purchased from Peprotech®).CD34+ cells were electroporated with the Cas9 RNP (Cas9 protein andms-sgRNA at a 1:1 weight ratio) using Lonza® 4D-Nucleofector™ and P3Primary Cell Kit (Program CA-137). For electroporation with dualms-sgRNAs, equal amount of each ms-sgRNA was added. Cells were culturedat 37° C. until analysis.

Genomic DNA Analysis

Genomic DNA was extracted from cells 2 days post electroporation usingprepGEM DNA extraction kit (ZyGEM™). Genomic region of interest wasamplified by PCR. PCR amplicons were analyzed by Sanger sequencing(Genewiz®) and allele modification frequency was calculated using TIDE(Tracking of Indels by Decomposition) software available on the WorldWide Web at tide.deskgen.com.

In Vitro Colony Forming Unit (CFU) Assay

Two days after electroporation, 500 CD34+ cells were plated in 1.1 mL ofmethylcellulose (MethoCult™ H4034 Optimum, Stem Cell Technologies™) on 6well plates in duplicates and cultured for two weeks. Colonies were thencounted and scored using StemVision™ (Stem Cell Technologies®).

Flow Cytometric Analysis and Sorting

Flurochrome-conjugated antibodies against human CD33 (P67.6), CD123(9F5), and CLL1 (REA431) were purchased from Biolegend®, BD Biosciences®and Miltenyi Biotec®, respectively. All antibodies were tested withtheir respective isotype controls. Cell surface staining was performedby incubating cells with specific antibodies for 30 min on ice in thepresence of human TruStain FcX™. For all stains, dead cells wereexcluded from analysis by DAPI (Biolegend®) stain. All samples wereacquired and analyzed with Attune® NxT flow cytometer (ThermoFisherScientific®) and FlowJo® software (TreeStar).

For flow cytometric sorting, cells were stained withflurochrome-conjugated antibodies followed by sorting with MoflowAstrios Cell Sorter (Beckman Coulter®).

CAR Constructs and Lentiviral Production

Second-generation CARs were constructed to target CD33, CD123, andCLL-1, with the exception of the anti-CD33 CAR-T used in CD33/CLL-1multiplex cytotoxicity experiment. Each CAR consisted of anextracellular scFv antigen-binding domain, using CD8α signal peptide,CD8α hinge and transmembrane regions, the 4-1BB costimulatory domain,and the CD3ξ signaling domain. The anti-CD33 scFv sequence was obtainedfrom clone P67.6 (Mylotarg®); the anti-CD123 scFv sequence from clone32716; and the CLL-1 scFv sequence from clone 1075.7. The anti-CD33 andanti-CD123 CAR constructs uses a heavy-to-light orientation of the scFv,and the anti-CLL1 CAR construct uses a light-to-heavy orientation. Theheavy and light chains were connected by (GGGS)3 linker. CAR cDNAsequences for each target were sub-cloned into the multiple cloning siteof the pCDH-EF1α-MCS-T2A-GFP expression vector, and lentivirus wasgenerated following the manufacturer's protocol (System Biosciences®).Lentivirus can be generated by transient transfection of 293TN cells(System Biosciences®) using Lipofectamine™ 3000 (ThermoFisher®). The CARconstruct was generated by cloning the light and heavy chain ofanti-CD33 scFv (clone My96), to the CD8α hinge domain, the ICOStransmembrane domain, the ICOS signaling domain, the 4-1BB signalingdomain and the CD3ξ signaling domain into the lentiviral plasmidpHIV-Zsgreen.

CAR Transduction and Expansion

Human primary T cells were isolated from Leuko Pak (Stem CellTechnologies™) by magnetic bead separation using anti-CD4 and anti-CD8microbeads according to the manufacturer's protocol (Stem CellTechnologies™). Purified CD4+ and CD8+ T cells were mixed 1:1, andactivated using anti-CD3/CD28 coupled Dynabeads™ (Thermo Fisher®) at a1:1 bead to cell ratio. T cell culture media used was CTS™ Optimizer™ Tcell expansion media supplemented with immune cell serum replacement,L-Glutamine and GlutaMAX™ (all purchased from Thermo Fisher®) and 100IU/mL of IL-2 (Peprotech®). T cell transduction was performed 24 hourspost activation by spinoculation in the presence of polybrene (Sigma®).CAR-T cells were cultured for 9 days prior to cryopreservation. Prior toall experiments, T cells were thawed and rested at 37° C. for 4-6 hours.

Flow Cytometry Based CAR-T Cytotoxicity Assay

The cytotoxicity of target cells was measured by comparing survival oftarget cells relative to the survival of negative control cells. ForCD33/CD123 multiplex cytotoxicity assays, wildtype and CRISPR/Cas9edited MOLM-13 cells were used as target cells, while wildtype andCRISPR/Cas9 edited HL60 cells were used as target cells for CD33/CLL-1multiplex cytotoxicity assays. Wildtype Raji cell lines (ATCC®) wereused as negative control for both experiments. Target cells and negativecontrol cells were stained with CellTrace™ Violet (CTV) and CFSE (ThermoFisher®), respectively, according to the manufacturer's instructions.After staining, target cells and negative control cells were mixed at1:1.

Anti-CD33, CD123, or CLL1 CAR-T cells were used as effector T cells.Non-transduced T cells (mock CAR-T) were used as control. For theCARpool groups, appropriate CAR-T cells were mixed at 1:1. The effectorT cells were co-cultured with the target cell/negative control cellmixture at a 1:1 effector to target ratio in duplicate. A group oftarget cell/negative control cell mixture alone without effector T cellswas included as control. Cells were incubated at 37° C. for 24 hoursbefore flow cytometric analysis. Propidium iodide (ThermoFisher®) wasused as a viability dye. For the calculation of specific cell lysis, thefraction of live target cell to live negative control cell (termedtarget fraction) was used. Specific cell lysis was calculated as((target fraction without effector cells—target fraction with effectorcells)/(target fraction without effectors))×100%.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

What is claimed is:
 1. An isolated population of cells comprisinggenetically engineered hematopoietic cells (HCs) or descendants thereof,wherein the genetically engineered HCs comprise (i) a geneticallyengineered gene encoding CD33 that is engineered to have reduced oreliminated expression of CD33, and (ii) a genetically engineered geneencoding CD123 that is engineered to have reduced or eliminatedexpression of CD123, and wherein the genetically engineered HCs aregenetically engineered using a CRISPR system comprising one or moreguide nucleic acids comprising a sequence according to SEQ ID NOs: 67,135, and/or 136, or a complement thereof.
 2. The isolated population ofcells of claim 1, wherein exon 3 of the genetically engineered geneencoding CD33 is genetically engineered.
 3. The isolated population ofcells of claim 1, wherein the genetically engineered gene encoding CD123and/or the genetically engineered gene encoding CD33 comprises aframeshift mutation.
 4. The isolated population of cells of claim 1,wherein the genetically engineered gene encoding CD123 and/or thegenetically engineered gene encoding CD33 is modified such that an exonis skipped.
 5. The isolated population of cells of claim 1, wherein thegenetically engineered gene encoding CD123 and/or the geneticallyengineered gene encoding CD33 comprises an indel mutation.
 6. Theisolated population of cells of claim 1, wherein exon 5 or exon 6 of thegenetically engineered gene encoding CD123 is genetically engineered. 7.The isolated population of cells of claim 1, wherein the expressionlevel of the genetically engineered gene encoding CD123 and/or thegenetically engineered gene encoding CD33 is less than 50% of theexpression level of the corresponding wild-type gene.
 8. The isolatedpopulation of cells of claim 1, wherein the genetically engineered HCshave reduced or no binding to an agent comprising an anti-CD33 bindingdomain and/or reduced or no binding to an agent comprising an anti-CD123binding domain.
 9. The isolated population of cells of claim 1, whereinat least 50% of the cells of the isolated population of cells aregenetically engineered HCs comprising the genetically engineered geneencoding CD33 and the genetically engineered gene encoding CD123. 10.The isolated population of cells of claim 1, wherein each cell of theisolated population of cells is a genetically engineered HC comprisingthe genetically engineered gene encoding CD33 and the geneticallyengineered gene encoding CD123.
 11. The isolated population of cells ofclaim 1, wherein the guide nucleic acid comprises a sequence accordingto SEQ ID NO: 67 or a complement thereof.
 12. The isolated population ofcells of claim 1, wherein the guide nucleic acid comprises a sequenceaccording to SEQ ID NO: 135 or a complement thereof, or SEQ ID NO: 136or a complement thereof.
 13. The isolated population of cells of claim1, wherein the genetically engineered HCs are genetically engineeredusing a CRISPR system comprising a guide nucleic acid comprising thesequence according to SEQ ID NO: 67, or complement thereof, and a guidenucleic acid comprising the sequence according to SEQ ID NO: 135, orcomplement thereof.
 14. The isolated population of cells of claim 1,wherein the genetically engineered HCs are genetically engineered usinga CRISPR system comprising a guide nucleic acid comprising the sequenceaccording to SEQ ID NO: 67, or complement thereof, and a guide nucleicacid comprising the sequence according to SEQ ID NO: 136, or complementthereof.
 15. The isolated population of cells of claim 1, wherein theCRISPR system comprises a Cas endonuclease.
 16. The isolated populationof cells of claim 1, wherein the CRISPR system comprises a Cas9endonuclease.
 17. The isolated population of cells of claim 1, whereinthe genetically engineered HCs comprise HCs obtained from a healthydonor, or descendant cells thereof.
 18. The isolated population of cellsof claim 1, wherein the genetically engineered HCs comprise HCs obtainedfrom bone marrow cells, cord blood cells, or peripheral bloodmononuclear cells (PBMCs), or descendants thereof.
 19. The isolatedpopulation of cells of claim 1, wherein the genetically engineered HCsare capable of expanding by at least 2-fold over 8 days in culture. 20.The isolated population of cells of claim 1, wherein the geneticallyengineered HCs retain the capacity to differentiate normally compared toa wild-type counterpart cell.
 21. The isolated population of cells ofclaim 1, wherein the genetically engineered HCs are capable ofengraftment.
 22. The isolated population of cells of claim 1, whereinthe genetically engineered HCs are CD34+ hematopoietic stem cells(HSCs).